The hepatic lipid peroxidation, copper and fibrosis in cholestatic rats

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THE HEPATIC LIPID PEROXIDATION, COPPER AND FIBROSIS IN CHOLESTATIC RATS GÜLÇIN AYKAC*t, GÜL ÖZDEMIRLER*, NILGÜN ALPTEKIN*, NEVIN ARICAN*, BOGE OZt, MÜJDAT UYSAL* and HIKMET OZ* *Department of Biochemistry, Istanbul Faculty of Medicine, University of Istanbul, Çapa, Istanbul, Turkey ; lDepartment of General Pathology, Cerrahpa~a Faculrv of Medicine, University of Istanbul, Cerrahpaca, Istanbul, Turkey Received in final form 15 April 1989

SUMMARY Hepatic lipid peroxidation was shown to be stimulated in the livers of cholestatic rats with increased hydroxyproline levels . In another group, cholestatic rats were fed with a copper-supplemented diet to increase hepatic copper levels . Although liver copper concentrations increased about 16-fold in copper supplemented cholestatic rats compared to normally fed cholestatic rats, no change was observed either in hepatic lipid peroxidation or in hydroxyproline levels . Krv woRos : lipid peroxidation, fibrosis, copper, cholestasis . INTRODUCTION Experimental cholestasis, known to stimulate collagen synthesis, is commonly used as a model for producing liver fibrosis and liver injury [1] . Besides fibrosis, hepatic copper accumulation is also known to be accompanied by prolonged cholestasis, since about 80% of absorbed copper is normally excreted into the bile [2] . Although the increased copper levels in cholestasis are not considered deleterious [3], it has been reported that lipid peroxidation led to liver injury in rats chronically treated with copper [4] . Therefore, in the present study, experimental chronic cholestasis is used as a model to examine the possible relations between the development of fibrosis, copper concentrations and lipid peroxidation in the livers of bile-duct ligated rats fed with or without copper-supplemented diet .

MATERIALS AND METHODS All reagents were analytical grade and were purchased from Merck, Darmstadt, FRG, except thiobarbituric acid and sodium dodecyl sulphate, purchased from tTo whom correspondence should be addressed . 1043-6618/89/060701-06/$03 .00/0

© 1989 The Italian Pharmacological Society



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Sigma Chemical Co. St . Louis, USA . Redistilled water was used throughout the work . Four groups of male Wistar rats weighing 200-250 g were used . Normal and bile-duct ligated rats were fed with normal diet for 15 days (groups 1 and 3, respectively) . In addition, normal and bile-duct ligated rats were fed with coppersupplemented diet, 2 g copper as CuSO 4 added per kg diet, for 15 days (groups 2 and 4, respectively) . Experimental cholestasis was induced by bile-duct ligation in ether anaesthetized rats according to Alemdaroklu et al. (groups 3 and 4) [5] . Groups 1 *and 2 were sham-operated . Two weeks after operation, animals were starved for 16 hours, killed by decapitation and their blood was collected for the determination of plasma total bile acids, total bilirubin, plasma glutamateoxalacetate transaminase (GOT), glutamate-pyruvate transaminase (GPT) and alkaline phosphatase (ALP) activities . Total bile acid determination was carried out by using commercial assay kits (Merckotest) . Total and conjugated bilirubin were measured according to Routh [6] and GOT, GPT and ALP were determined by Technicon RA 1000 selective autoanalyser . The livers were removed and slices from the left, right and median lobes of liver were fixed in 10% neutral formol solution for histological studies . The remaining part of liver was used for hydroxyproline, lipid peroxidation and copper determinations . Total liver hydroxyproline was assayed as a marker of fibrosis according to Bergman and Loxley [7] : 1 ml of 20% liver homogenate in acetate/citrate buffer, pH 6 . 0 was hydrolysed in 1 ml 6 M HCI . After neutralization 0 . 5 ml isopropanol and 0 . 25 ml oxidant solution (equal volumes of 7% chloramine T and acetate/ citrate buffer, pH 6 . 0) were added to 0 . 25 ml of hydrolysed sample, well mixed and allowed to stand for 4 min at room temperature . 3 . 25 ml of Ehrlich reagent (2 g of p-dimethylaminobenzaldehyde was dissolved in 60% perchloric acid . To 3 volumes of this solution, 13 volumes of isopropanol were added to get the final Ehrlich reagent) were added, mixed and the tubes heated for 25 min at 60°C . After cooling, absorbances were read at 558 nm against water. Hydroxyproline was used as standard . Hepatic copper levels were determined by atomic absorption spectrophotometer in liver homogenates [8] : 5 ml of 10% liver homogenate were dried at 90°C and wet-ashed by further heating to 200-210°C with 5 ml of a mixture of 70% HC1O 4 (perchloric acid, Merck Suprapur) and 65% nitric acid (Merck Suprapur) (1 :1) . The white residue was dissolved in 5 ml 6 M HNO 3 and measurements were performed with a Perkin-Elmer flameless atomic absorption spectrophotometer . Standard copper solution standardized for atomic absorption was used (Hartman-Leddon company, Philadelphia, USA) . Hepatic lipid peroxidation was carried out by determining malondialdehyde (MDA) levels as described by Ohkawa et al. [9] : 0 . 2 ml 8 . 1% sodium dodecyl sulphate, 1 . 5 ml 20% acetic acid solution (pH 3 . 5) and 1 . 5 ml 0 . 8% aqueous solution of thiobarbituric acid (TBA) were added to 0 . 1 ml 10% (w/v) liver homogenate in 0 . 15 M KC1. The mixture was made up to 4 .0 ml with H,O and heated at 95°C for 60 min. After cooling, 1 . 0 ml water and 5 . 0 ml of the mixture of n-butanol/pyridine (15 :1, v/v) were added and shaken vigorously . After



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centrifugation, the absorption of organic layer was measured at 532 nm . To eliminate the interference of bilirubin, fluorometric measurements (excitation 515 nm, emission 553 run) were also carried out . 1,1,3,3-Tetraethoxypropane was used as standard . Protein determinations were accomplished by the method of Lowry et al. [ 10 J .

Table I Total bile acid, bilirubin levels, GOT, GPT and ALP activities in the plasma and hydroxyproline, lipid peroxide and copper levels in the livers of normal and bile-duct ligated rats fed with or without copper-supplemented diet for 15 days Parameter Plasma Total bile acids µmol/l ) Total bilirubin (mg/dl ) GOT (units/I) GPT (units/1) ALP (IU/I) Liver Hydroxyproline (ug/g wet tissue) Lipid peroxide (pmol MDA/mg protein) Copper (pg/g wet tissue)

Group I

Group 2

5 . 00±2 . 10

5 . 10±1 . 95

Group 3

89 . 75±37 . 3*

Group 4

93 . 3±27 . 1*

0 . 50±0 . 05 125 . 0 ± 20 . 8 45 . 0±7 . 70 187 . 2 ± 57 . 5

0 . 54±0 . 07 5 . 13±3 . 47* 4 . 96±2 . 84* 454-4±86-8* 480-1±128-2* 572-4+140-2* 135 . 0±28 . 0* 156 . 2±62 . 2* 163 . 6±59 . 1* 190 . 0 ± 45 . 3 443-6±228-4** 340-7± 102 . 5

134 . 1 ± 23 . 1

125 . 1 ± 16 . 7

247 . 5 ± 41 . 9*

550 . 3 ± 55 . 3

542 . 6 ± 64 . 1

832-8±128-1* 860-2± 137 . 2*

269 . 7 ±62-4*

17 . 70 ±3-40*

4 . 37 ± 0 . 78

263 . 7± 59 . 0*

299 . 3 ± 42 . 9*

Values represent means and their standard deviations (X± SD, n= 8 each). Group 1, normal rats fed with normal diet ; group 2, normal rats fed with coppersupplemented diet ; group 3, bile-duct ligated rats fed with normal diet ; group 4, bile-duct ligated rats fed with copper-supplemented diet . *P< 0 . 001, **P<0-01 as compared to controls ; Student's t-test . RESULTS Cholestasis formation induced by bile-duct ligation was shown by increases in plasma ALP activity, total bile acid and bilirubin levels, where elevated plasma GOT and GPT activities were used as indices of marked cholestatic liver damage in group 3 and 4 (Table I) . Pathological examinations revealed the presence of severe portal fibrosis, pericholangitis, bile-duct regeneration and biliary cirrhosis in the livers of cholestatic rats in both groups 3 and 4, having almost similar appearances . Plasma GOT and GPT activities were observed to be affected by copper supplementation to normal rats, but no histopathological changes could be detected other than hydropic swelling of parenchymal cells (group 2) (Figs I and 2) .



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Fig . 1 . Livers of cholestatic rats in groups 3 and 4 . Portal fibrosis, pericholangitis, biliary cirrhosis (haematoxylin-eosin x 80) .

Fig. 2 . Livers of cholestatic rats in groups 3 and 4 . Portal fibrosis, bile-duct regeneration, inflammatory reaction (haematoxylin-eosin x 200) .



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Parallel to histological findings, hydroxyproline levels rose significantly, indicating an accumulation of collagen in the livers of the animals of groups 3 and 4 compared to 1 . In addition, hepatic lipid peroxide levels increased significantly in the same groups compared to group I (Table I) . No difference could be detected in hepatic hydroxyproline or lipid peroxide levels of the animals of group 2 compared to group 1 (Table I) . We found approximately a 70-fold increase in hepatic copper levels of group 4, where in group 3 it was four-fold and in group 2 it was 60-fold, compared to group 1 (Table I) . To summarize : 1 . Experimental cholestatis caused increases in hepatic hydroxyproline and lipid peroxide levels with hepatic copper concentration increasing moderately (fourfold) in normal fed bile-duct ligated rats, 2 . Although copper concentration increased about 70-fold, copper supplementation to cholestatic rats seemed to be ineffective in increasing hepatic hydroxyproline and lipid peroxide levels when compared to those of cholestatic rats fed with normal diet . 3 . Copper supplementation to normal rats had no effect either on hepatic lipid peroxidation or on hydroxyproline levels, although hepatic copper concentrations increased 60-fold compared to normal fed rats .

DISCUSSION Synthesis and extent of collagen fibre deposits have a great influence on the course and prognosis of chronic liver disease [11] . Under physiological conditions, the total collagen content of the liver is low, where rates of synthesis and breakdown are in a steady state [12] . Cholestasis is an example in which this steady state is disturbed on the behalf of synthesis [1] . The mechanism and the cause of the stimulation in collagen synthesis in cholestasis is not clear . In a recent study, superoxide anion production was shown to be stimulated in human polymorphonuclear neutrophils incubated with collagen [ 13] . In another study, on the other hand, collagen synthesis was shown to be stimulated in hepatocytes exposed to superoxide radicals [ 14] . For this reason, it may be suggested that one of the factors affecting the rate of collagen synthesis in cholestasis is the presence of free radicals in the medium . Indeed, in this study, the increased levels of hepatic hydroxyproline, a reliable parameter of hepatic collagen synthesis, were shown to be in conformity with hepatic lipid peroxide levels . Thus, it may be proposed that increase in collagen synthesis causes the stimulation of superoxide formation and thus lipid peroxidation, or that the increase in lipid peroxidation is an indication of increased amounts of free radicals which cannot he eliminated by natural defence systems and thus may lead to the stimulation of collagen synthesis . On the other hand, the role of copper in the stimulation of collagen synthesis and cholestatic injury is not clear . In a previous study, Dillard and Tappel showed the stimulation of hepatic lipid peroxidation in rats chronically treated with copper [4', . In contrast, Barka et al. could not observe either liver cirrhosis or accumulation



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of chromolipids in rats after 81 days of daily injections of copper

[15] . These

conflicting results may arise from the species differences, the type, the dose and the duration of copper loading [16] . In addition, the site of copper deposited in the cell plays an important role in its toxicity [3] . In our work, to detect the relation between copper, hepatic hydroxyproline and lipid peroxidation levels, cholestatic rats were fed with or without copper supplemented diet and no difference could be observed either in hydroxyproline or in lipid peroxide levels by increasing hepatic copper concentration about 16-fold with copper supplementation (group 4 compared to group 3) . In conclusion, it can be suggested that hepatic lipid peroxidation and liver collagen synthesis might be interlinked events and increased copper concentrations in the livers of cholestatic rats may be considered not responsible for the increases in hepatic levels of hydroxyproline and lipid peroxidation .

REFERENCES 1 . Oelberg DG, Lester R. Cellular mechanisms of cholestatis . Ann Rev Med 1986 ; 37 : 297-317 . 2 . Evans J, Newman S, Sherlock DS . Liver copper levels in intrahepatic cholestasis of childhood . Gastroenterology 1978 ; 75 : 875-8 . 3 . Sherlock DS . Diseases of the liver and the biliary system . 7th ed . Oxford : Blackwell Scientific Publications, 1985 : 222 . 4 . Dillard CJ, Tappel AL . Lipid peroxidation and copper toxicity in rats . Drug Chem Toxicol 1984 ; 7 : 477-87 . 5 . Alemdaroglu K, Gürakar M, Ozbal A, Sinn F, Büyükünal C . Peritonevenous shunts . Cerrahpa4a Tip Fak Derg 1980 ; 11 : 162-7 . 6 . Routh JI. Liver function . In: Tietz NW, ed . Fundamentals of clinical chemistry . Philadelphia: WB Saunders, 1976 :1026-62 . 7. Bergman I, Loxley R. Two improved and simplified methods for the spectrophotometric determination of hydroxyproline . Anal Chem 1961 ; 35 : 1961-3 . 8 . Wittig M, Steffen C . Modification of microsomal lipid peroxidation and drug metabolism by cytoplasmic copper. Res Commun Chem Pathol Pharmacol 1984 ; 44 :477-93 . 9 . Ohkawa H, Ohishi N, Yagi K . Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction . Anal Biochem 1979 ; 95 : 351-8 . 10. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ . Protein measurements with the folin phenol reagent . J Biol Chem 1951 ; 193 : 265-75 . 11 . Müller A, Machnic F, Zimmerman T, Shubert H . Thioacetamide-induced cirrhosis-like liver lesions in rats-usefulness and reliability of this animal model . Exp Pathol 1988 ; 34 : 229-36 . 12. Gressner AM . Fibroplasie der Leber-aktueller Kenntnisstand molekularer and zellulärer Mechanismen . Med Welt 1986 ; 37 : 898-905 . 13 . Monboisse JC, Bellon G, Dufer J, Randoux A, Borel J . Collagen activates superoxide anion production by human polymorphonuclear neutrophils . Biochem J 1987 ; 246 : 599-603 . 14. Hussain MZ, Watson JA, Bhatnager RS . Increased prolyl-hydroxylase activity and collagen synthesis in hepatocyte cultures exposed to superoxide . Hepatology 1987 ; 7 : 502-7 . 15 . Barka T, Scheuer PJ, Schaffner F, Popper H . Structural changes in liver cells in copper intoxication . Arch Pathol 1964 ; 78 : 331-49 . 16 . Haywood S . The effect of excess dietary copper on the liver and kidney of the male rat . J Comp Path 1980 ; 90: 217-32 .