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Does oxidative protein damage play a role in the pathogenesis of carbon tetrachloride-induced liver injury in the rat?
Biochimica et Biophysica Acta 1362 Ž1997. 169–176
Does oxidative protein damage play a role in the pathogenesis of carbon tetrachloride-induced liver injury in the rat? Premila N. Sundari a
a,)
, Wilfred G a , Banumathi Ramakrishna
b
Department of Biochemistry, Christian Medical College & Hospital, Vellore 632002, Tamilnadu, India Department of Pathology, Christian Medical College & Hospital, Vellore 632002, Tamilnadu, India
b
Received 28 July 1997; accepted 13 August 1997
Abstract Free radicals have been implicated in the pathogenesis of alcohol-induced liver injury in humans and carbon tetrachloride ŽCCl 4 .-induced liver injury in rats. The most extensively studied aspect of free radical induced liver injury is lipid peroxidation. Recently it has been found that free radicals can cause oxidative damage to cellular proteins and alter cellular function. One such susceptible protein is the enzyme glutamine synthase ŽGS.. The chemical effects of CCl 4 on cell proteins and their biological consequences are not known. Hence, in our study, the effect of CCl 4 on liver protein oxidation and GS activity were investigated and compared with lipid peroxidation. A significant increase in liver protein carbonyl content Ž2–3 fold. and a significant decrease in hepatic GS activity Ž44–57%. were observed. Damage to proteins was rapid in onset and increased with time. Acute exposure of rats to CCl 4 resulted in an increase in hepatic protein carbonyl content and a decrease in hepatic GS within 1 h. In cirrhosis of the liver induced by CCl 4 , the decrease in hepatic GS activity was accompanied by a significant increase in plasma ammonia levels. We conclude that protein oxidation may play a role in the pathogenesis of CCl 4 induced liver injury and that the accumulation of oxidised proteins may be an early indication of CCl 4 induced liver damage. q 1997 Elsevier Science B.V. Keywords: Carbon tetrachloride; Liver injury; Protein oxidation; Glutamine synthase; Lipid peroxidation
1. Introduction Cirrhosis of the liver is one of the major health problems in developed countries. Cirrhosis of the liver is the fourth leading cause of death in the American adults w1x, about 70% of which is associAbbreviations: CCl 4 , carbon tetrachloride; GS, glutamine synthase; PCo, protein carbonyl; LPo, lipid peroxide; MDA, malondialdehyde; TCA, trichloroacetic acid; DNPH, 2,4-dinitrophenylhydrazine; HEPES, 4-Ž2-hydroxyethyl. 1-piperazine ethane sulphonic acid; PV, perivenular; AST, aspartate aminotransferase; ALT, alanine aminotransferase ) Corresponding author. Tel.: 91-416-22603-4267; fax: 0091416-32788; E-mail: [email protected]
ated with alcoholism. Although extensive work is being done in the field of cirrhosis, the molecular mechanisms leading to cell death and increased collagen accumulation are still not clear. Therefore, treatment and prevention of the fibrotic process is still elusive. The most widely accepted animal model of cirrhosis is the ratrcarbon tetrachloride ŽCCl 4 . model, which is remarkably similar to human alcoholic cirrhosis both histologically w2,3x and systemically w4,5x. The involvement of free radicals is reported in both cases. So far, the most extensively investigated mechanism of free radical induced liver injury is lipid peroxidation w6–9x. Only recently, the possibility that free radicals may cause potential damage to proteins
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P.N. Sundari et al.r Biochimica et Biophysica Acta 1362 (1997) 169–176
has been taken into account and a wide range of changes have been reported w10–12x. The formation of carbonyl derivatives of proteins is suggested to be a useful measure of oxidative damage to proteins. Key metabolic enzymes have been shown to be inactivated by oxygen free radicals produced by either mixed function oxidase system or by non-enzymatic methods w13x. The oxidative inactivation of enzymes by free radicals and the intracellular accumulation of oxidised proteins may play a critical role in the alteration of cellular function and cell death. The chemical effects of CCl 4 on cell proteins and the biological consequences of such reactions have not been studied. CCl 4 is metabolically activated to free radicals mainly in perivenular ŽPV. hepatocytes which are rich in the drug metabolising microsomal cytochrome P450 system w14x. GS which plays a pivotal role in nitrogen metabolism, including detoxification of ammonia is found exclusively in the PV hepatocytes. It is in fact considered to be a PV marker enzyme w15,16x. Exposure of GS to non-enzymatic mixed function oxidase system composed of ascorbate, oxygen and FeŽIII. has been shown to result in the oxidative modification of the enzyme. Oxidative modification of GS abolishes its catalytic activity w17,18x. Due to the co-localization of cytochrome P450 and GS on the microsomes of PV hepatocytes, it is likely that free radicals generated from CCl 4 by cytochrome P450 may cause oxidative inactivation of GS. The objectives of the present study were to find out Ž 1. whether oxidation of liver proteins occurs in acute and chronic CCl 4 administration; Ž2. whether there is any alteration in hepatic GS activity; and Ž 3. to find out whether there is any relationship between hepatic GS and plasma ammonia levels. Our results provide evidence for liver protein oxidation and also for a link between liver protein carbonyl ŽPCo. content and the activity of hepatic GS.
vanised iron cages and maintained on standard laboratory rat chow Žsupplied by Lipton India. with free access to tap water in a thermostatically controlled room Ž288C. under 12 h darkrlight cycle.
2. Materials and methods
2.3.2. Acute exposure to CCl 4 The rats were administered phenobarbitone in drinking water Ž500 mgrl. for 10 days. The rats were then exposed to CCl 4 vapours once for a period of 10 min as described above. The rats were sacrificed at
2.1. Animals Adult male Wistar rats weighing 150–200 g were used for the study. The rats were housed in gal-
2.2. Chemicals 2,4-Dinitrophenyl hydrazine, protease inhibitors Žleupeptin, pepstatin A, aprotinin and phenylmethylsulphonyl fluoride., HEPES buffer, ATP, g-glutamyl hydroxamate, 1,1,3,3-tetramethoxy propane and glutamate dehydrogenase were obtained from Sigma, St. Louis, MO, USA. All other reagents were of the highest purity available. 2.3. Animal treatment 2.3.1. Induction of cirrhosis Cirrhosis was induced in the rats by chronic exposure to CCl 4 vapours according to the method of McLean et al. w5x. A wooden box, with a glass front Ž72 = 45 = 46 cm, i.e., 150 l capacity. was fitted with an inlet tap and housed in a fume cupboard. Compressed air was passed via a flow meter, at 4 lrmin, bubbling through a train of two wash bottles containing ‘‘Analar’’ CCl 4 maintained at 208C, and into the box. Air left the box by leaking through the joints. CCl 4 was run in for 5 min and was then turned off and the rats left in the box for 5 more minutes Žtherefore, total period of 10 min.. Treatment was carried out twice a week for 12 consecutive weeks. Tap water containing phenobarbitone Ž500 mgrl. was the only source of drinking water for the rats 10 days prior to the first dose of CCl 4 and throughout the treatment period. At the end of 12 weeks, the rats were sacrificed. Slices of liver tissue were fixed in 10% formalin and processed and stained with haematoxylin–eosin, Foot’s reticulin and Van Gieson stains for histological assessment. The time period of 12 weeks was found to be sufficient to induce cirrhosis in the rats. Control rats were kept under the same conditions as the experimental rats, but were administered phenobarbitone alone.
34.00 " 8.81 Žns . Ž n s 6 . 72.8 " 5.76 Žns . Ž n s 5 . 23.17 " 5.08 Žns . Ž n s 6 . 65.25 " 4.57 Žns . Ž n s 4 . 20.67 " 3.27 Ž n s 6 . 65.67 " 6.86 Ž n s 6 .
Data represent mean value " SD. a P - 0.001; b P - 0.01; c P - 0.05. ns – statistically not significant compared with zero time, nd – not determined, n s no. of rats. Rats were sacrificed at various time intervals after exposure to CCl 4 vapours for 10 min. Liver was excised, homogenised and assays carried out as described in Section 2. Protein carbonyl content was calculated from extinction co-efficient of 21.0 mMy1 cmy1 for aliphatic hydrazones. One unit of GS activity is defined as the amount of enzyme that catalyses the formation of 1 m mol of gamma glutamyl hydroxamate per hour at 378C.
nd
68.75 " 19.56 Ž n s 4 . 64.25 " 10.87 c Ž n s 4 . 94.5 " 10.38 Žns . Ž n s 4 . 66.50 " 5.2 Žns. Ž n s 4 . 121.2 " 25.41 Ž n s 5 . 183.2 " 24.44 c Ž n s 5 . 85.25 " 29.32 Ž n s 4 . 89.00 " 20.94 Žns . Ž n s 4 .
c
98.57 " 6.9 Žns . Ž n s 8 .
c
nd nd nd 92.95 " 6.5 Ž n s 6 .
0.54 " 0.08 a Ž n s 6 . 0.60 " 0.04 b Ž n s 5 . 0.86 " 0.14 Ž n s 6 .
nd
c
0.48 " 0.04 a Ž n s 6 . nd 0.47 " 0.09 a Ž n s 5 .
393.6 " 132.8 b Ž n s 5 . 267.7 " 48.8 Žns. Ž n s 5 . 252.8 " 82.6 Ž n s 6 .
0.49 " 0.09 a Ž n s 5 .
822.1 " 207.3 a Ž n s 4 . 1139.6 " 192.5 a Ž n s 4 . 1877.1 " 75.6 a Ž n s 4 .
18 7.76 " 0.41 a Ž n s 3 .
14 8
nd 7.68 " 0.83 a Ž n s 6 .
4
6.53 " 0.36 a Ž n s 6 .
2
5.05 " 0.28 Ž n s 6 . 0
171
Protein carbonyl content Žnmolrmg protein . Lipid peroxide level Žnmolrg wet weight. Glutamine synthase activity ŽUrmg protein . Plasma ammonia level Ž m g% . Plasma ALT ŽUrl. Plasma AST ŽUrl.
Time Žh . after a single dose of CCl 4
2.4.2. LiÕer protein carbonyl content About 250 mg of liver was minced and homogenised in 10 ml of ice-cold 10 mmolrl HEPES buffer, pH 7.4 containing 125 mmolrl KCl, 5 mmolrl Na 2 EDTA and protease inhibitors wleupeptin Ž0.5 mgrl. , pepstatin Ž 0.7 mgrl. , aprotinin Ž 0.5 mgrl. and phenylmethylsulphonyl fluoride Ž40 mgrl.x to inhibit protease activities. PCo content was measured as described by Levine et al. w20x. Aliquots of homogenate were precipitated with 20% trichloroacetic acid ŽTCA. . The pellet was suspended in 10 mmolrl DNPH in 2 molrl HCl and allowed to stand at room temperature for 1 h, with vortexing every 10 min. Proteins were then reprecipitated with 20% TCA and centrifuged. The pellet was washed thrice with 1 ml of ethanolrethyl acetate Ž1 : 1 vrv.. The final pellet was dissolved in 1 ml of 6 molrl guanidine hydrochloride Žin 20 mmolrl potassium phosphate buffer pH 2.3.. The difference in absorbance of the sample treated with DNPH in HCl was determined versus the sample treated with HCl alone at 366 nm. Results are expressed as nmoles of DNPH incorporatedrmg of
Parameter
2.4.1. Plasma ammonia Plasma ammonia was estimated enzymatically using glutamate dehydrogenase and the change in absorbance of NADPH was monitored spectrophotometrically at 340 nm w19x.
Table 1 Effect of a single dose of CCl 4 on hepatic protein carbonyl content, lipid peroxide level, glutamine synthase activity, plasma ammonia level and transaminases
The CCl 4 treated and the control rats were fasted overnight before sacrifice. Blood was collected from the animals under light ether anaesthesia by heart puncture into tubes containing Na 2 EDTA Ž 1 mgrml. and kept in ice. Plasma was separated immediately and used for the assay of ammonia and transaminases ŽALT, AST. . The livers were excised quickly, weighed and used for biochemical and histological studies. All the assays were carried out within 48 h of sacrifice of the animals.
8.66 " 0.95 a Ž n s 6 .
2.4. Biochemical assays
394.7 " 141.8 c Ž n s 6 .
28
the end of various time intervals Ž2, 4, 8, 14, 18 and 28 h.. In another experiment, the rats were exposed to a low dose of CCl 4 Žfor 6 min instead of 10 min. and sacrificed after 1 h. Phenobarbitone treated rats served as controls.
9.61 " 0.28 a Ž n s 4 .
P.N. Sundari et al.r Biochimica et Biophysica Acta 1362 (1997) 169–176
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P.N. Sundari et al.r Biochimica et Biophysica Acta 1362 (1997) 169–176
Fig. 1. Correlation between hepatic PCo content and specific activity of GS. The regression equation is y s 1.324 y 0.0929 x Ž r s y0.847, P - 0.001..
Fig. 2. Time dependent changes in PCo content, LPo level and GS activity in rat liver after a single dose CCl 4 .
protein calculated from an extinction coefficient of 21 mMy1 cmy1 for aliphatic hydrazones w21x.
2.4.5. Plasma transaminases Plasma aspartate aminotransferase Ž AST. and alanine aminotransferase ŽALT. activities were assayed as described by Bergmeyer and Bernt w25x.
2.4.3. Hepatic glutamine synthase actiÕity About 1 g of liver was minced and homogenised in 10 ml of ice-cold HEPES buffer pH 7.4 containing 125 mmolrl KCl and 5 mmolrl Na 2 EDTA. The homogenate was sonicated for 30 s at 200 V ŽVibronics, India.. The homogenate was centrifuged for 15 min at 16,000 = g at 48C and the supernatant was used for the determination of GS activity and protein. GS activity was assayed by the method described by Rowe et al. w22x. g-glutamyl hydroxamate was used as the standard. One unit of enzyme activity is defined as the amount of enzyme catalysing the formation of 1 mmol of g-glutamyl hydroxamate per hour at 378C.
3. Statistical analysis All parameters are expressed as mean values " S.D. Differences between mean values of tests and controls were evaluated statistically by Student’s t-test. A P value of - 0.05 was regarded as
Table 2 Hepatic protein carbonyl content, glutamine synthase activity and plasma transaminase levels 1 h after exposure of rats to low dose of CCl 4 vapours Parameter
2.4.4. Hepatic lipid peroxide leÕels One gram of liver was homogenised in 9 ml of 1.15% ice-cold KCl. Lipid peroxide ŽLPo. levels were measured in whole homogenate by the thiobarbituric acid reaction w23x. Results are expressed as nmoles of malondialdehyde Ž MDA. per gram wet weight of liver. Protein was measured by the method described by Lowry et al. using bovine serum albumin as standard w24x.
Hepatic PCo content Žnmolrmg protein. Hepatic GS activity ŽUrmg protein. Plasma ALT ŽUrl. Plasma AST ŽUrl.
Control rats Ž ns6.
CCl 4 treated rats Ž ns 7.
5.10 " 0.16
6.22 " 0.44 a
0.88 " 0.11
0.72 " 0.10 b
19.71 " 4.54 68.17 " 8.42
20.43 " 4.72 Žns. 70.57 " 7.66 Žns.
n s no. of rats. Data represent mean value " SD. P - 0.001; b P - 0.05. Rats were sacrificed 1 h after exposure to CCl 4 vapours for 6 min and assays carried out as described in Section 2. a
P.N. Sundari et al.r Biochimica et Biophysica Acta 1362 (1997) 169–176
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Fig. 3. Rat liver showing normal appearance and no necrosis 1 h after exposure to low dose of CCl 4 vapours. PT – portal tract, ThV – terminal hepatic vein. The stain used was hematoxylin–eosin Ž=90..
Fig. 4. Liver cirrhosis in rats treated with CCl 4 for 12 weeks. The stain used was Foot’s reticulin Ž=40..
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P.N. Sundari et al.r Biochimica et Biophysica Acta 1362 (1997) 169–176
Table 3 Hepatic protein carbonyl content, lipid peroxide level, glutamine synthase activity and plasma ammonia level in CCl 4-induced cirrhosis of the liver Parameter
Control
Cirrhosis
Protein carbonyl content Žnmolrmg protein. Lipid peroxide level Žnmolrg wet weight. GS activity ŽUrmg protein. Plasma ammonia level Žmgr100 ml.
4.12 " 0.39 Ž n s 6. 379.3 " 36.5 Ž n s 7. 1.00 " 0.21 Ž n s 8. 122.6 " 26.3 Ž n s 6.
11.19 " 1.02 a 551.3 " 92.5 b 0.43 " 0.10 a 262.5 " 77.6 b
Ž n s 8. Ž n s 8. Ž n s 10. Ž n s 10.
n s no. of rats. Data represent mean value " SD. P - 0.001; b P - 0.01. Rats were sacrificed after 12 weeks of treatment with CCl 4 as described in Section 2.
a
statistically significant. Karl Pearson’s test w26x was used for correlation studies.
4. Results 4.1. Acute exposure to CCl 4 Table 1 shows the time dependent effect of single dose of CCl 4 on hepatic PCo content, LPo level, GS activity, plasma ammonia level and transaminase levels. A significant increase in PCo content was observed Žapproximately 2 fold-28 h value. . This was accompanied by a significant decrease in GS activity Ž44%.. A clear significant inverse correlation be-
tween PCo content and GS activity was observed as shown in Fig. 1 Ž r s y0.847, P - 0.001. . A significant increase in hepatic MDA levels was also observed Ž7 fold-14 h value. . Fig. 2 shows time dependant changes in the above mentioned parameters. Liver PCo began to rise significantly as early as 2 h and showed an increase with time. Coincidentally, hepatic GS activity also began to fall at 2 h and remained low for 28 h. MDA level showed significant elevation at 4 h, peaking at 14 h and declining thereafter. There was no significant difference in plasma ammonia levels between the CCl 4 treated rats and the untreated rats. Plasma transaminases began to rise at 8 h, peaked at 14 h and began to decrease thereafter suggesting that hepatic necrosis had occurred to a certain extent in the later hours. In the rats sacrificed 1 h after the low dose of CCl 4 , the hepatic PCo level was increased by 18% and the activity of GS was decreased by 18% as shown in Table 2. There was no evidence of necrosis histologically as shown in Fig. 3. Plasma ALT and AST were unaltered. 4.2. Chronic liÕer injury
Fig. 5. Relationship between the specific activity of hepatic GS and plasma ammonia concentration in cirrhotic rats. The regression equation y s 522.815 y 661.997x Ž r s y0.819, P 0.05..
The rats that were exposed to CCl 4 vapours for 12 weeks developed cirrhosis as shown in Fig. 4. Table 3 shows the effect of chronic exposure of rats to CCl 4 on liver PCo, LPo, GS and plasma ammonia level. Nearly 3 fold increase in PCo was observed. GS activity in the cirrhotic rats was decreased significantly Ž57%.. A moderate, but significant increase in MDA levels Ž1.5 fold. was also observed in the cirrhotic rats. Plasma ammonia levels were elevated more than 2 fold in the cirrhotic animals and they
P.N. Sundari et al.r Biochimica et Biophysica Acta 1362 (1997) 169–176
showed significant inverse correlation with hepatic GS activity as shown in Fig. 5 Ž r s y0.819, P 0.05..
5. Discussion It is a well established fact that lipid peroxidation plays a major role in CCl 4 induced development of fatty liver and cirrhosis. Accordingly, we found significant increase in the MDA levels Ž7 fold-14 h value. upon single exposure to CCl 4 vapours. The decrease in MDA levels after 14 h may be due to the metabolism of MDA by the rat liver mitochondria w27x. A significant increase in the PCo content of the liver Ž approximately 2 fold-28 h value. was also observed. Nearly 2 fold increase in liver PCo after single dose of CCl 4 may not appear significant. However, it has been estimated that values as low as 2 nmol of carbonyl per mg protein represents damage to about 10% of total cellular proteins w28x Žthese are minimal values as the oxidative modification of some amino acid residues in protein does not lead to the formation of carbonyl derivatives. . Extrapolating from this, we could calculate that, in CCl 4 treated rats about 23% of total hepatocellular proteins are damaged. As mentioned earlier, we found significant increase in PCo levels even within an hour of exposure to CCl 4 vapours. This suggests that the accumulation of oxidised proteins in the liver may be an early indication of CCl 4 induced liver damage. Decrease in hepatic GS activity was observed upon single exposure to CCl 4 vapours. GS is present exclusively in the PV hepatocytes and is highly susceptible to oxidative damage by free radicals. The decrease in GS activity in acute CCl 4 administration may be due to the necrosis of PV hepatocytes or due to the oxidative inactivation of the enzyme. An increase in PCo content and a decrease in GS of the liver in the absence of necrosis would suggest oxidative inactivation of the enzyme. We found a significant increase in PCo levels and a significant decrease in GS in the liver 1 h after exposure to CCl 4 vapours Ž Table 2. , when there was no evidence of hepatocellular necrosis both histologically and biochemically. Therefore, decreased GS activity in acute CCl 4 administration can be attributed to the oxidative inac-
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tivation of the enzyme by trichloromethyl radical and trichloromethylperoxy radical generated from CCl 4 . Although significant decrease in hepatic GS was observed in acute exposure to CCl 4 vapours, plasma ammonia levels were unaltered. This could be explained on the basis of the fact that the other important enzymes concerned with the detoxification of ammonia namely the urea cycle enzymes are mainly periportal in their location w29x and are reported to be unaffected by CCl 4 , the PV toxin in short term exposure of rats w30x. Regarding chronic liver injury, a greater increase in liver PCo was observed Ž approximately 3 fold. compared to acute liver injury Ž approximately 2 fold.. As mentioned earlier values as low as 2 nmolrmg protein represents damage to about 10% of total cellular proteins. Extrapolating from this in CCl 4 treated rats about 31% of total hepatocellular proteins are damaged. A 1.5 fold increase in MDA levels was also observed. Such a low value in MDA levels may be due to the metabolism of MDA by the rat liver mitochondria as mentioned earlier. In chronic liver injury, the decrease in hepatic GS can be attributed mainly to the loss of functional hepatocytes. The significant inverse correlation between plasma ammonia levels and hepatic GS in the cirrhotic rats suggests that the decrease in the activity of hepatic GS may contribute to hyperammonemia observed in the cirrhotic animals, while other factors such as portosystemic shunting play important roles. A similar observation has been made earlier by Gebhardt and Reichen w31x, although they found a greater decrease in GS activity Ž80%. compared to our study Ž57% reduction.. These authors have also suggested that the decrease in GS activity in cirrhosis of the liver contributes to hyperammonemia. Our study suggests that oxidative damage to proteins occurs in acute as well as chronic exposure of rats to CCl 4 and that it may contribute to the pathogenesis of liver injury, while lipid peroxidation plays a major role. Our results also suggest that the accumulation of oxidised proteins in the liver may be an early indication of CCl 4 liver injury and that the decrease in hepatic GS activity Ž in the absence of hepatocellular necrosis. may be due to the oxidative inactivation of the enzyme. The decrease in GS activity in chronic liver injury induced by CCl 4 can be attributed to the loss of functional hepatocytes. The
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decrease in GS may contribute to hyperammonemia observed in cirrhotic animals, while factors such as portosystemic shunting play important roles. Acknowledgements This work was supported by the grants from the FLUID Research Fund, Christian Medical College & Hospital, Vellore-4. We are grateful to Mr. Tamilselvan for his technical assistance. References w1x S. Sherlock, J. Dooley, Diseases of the Liver and Biliary System, 9th ed., Blackwell Scientific Publications, Oxford, 1993, p. 370 w2x R.J. Stenger, Arch. Pathol. 81 Ž1966. 439–447. w3x E. Rubin, H. Popper, Medicine 46 Ž1967. 163–183. w4x P.M. Daniel, M.M.L. Prichard, P.C. Reynell, J. Pathol. Bacteriol., 64 Ž1952a. 53–60 w5x E.K. McLean, A.E.M. McLean, P.M. Sutton, Br. J. Exp. Pathol. 50 Ž1969. 502–506. w6x E. Albano, A. Tomasi, L. Goria-Gatti, M.U. Dianzani, in: C. Rice Evans ŽEd.., Free Radicals, Cell Damage and Disease, Richelieu Press, London, 1986, pp. 117–126 w7x E. Albano, A. Tomasi, L. Goria-Gatti, M.U. Dianzani, Chem. Biol. Interact. 65 Ž1988. 223–234. w8x E. Albano, A. Tomasi, J.O. Persson et al., Biochem. Pharmacol. 41 Ž1991. 1895-1902 w9x I. Johansson, G. Ekstrom, B. Scholte, D. Puzycky, H. Jornall, M. Ingelman-Sundberg, Biochemistry 27 Ž1988. 1925–1934. w10x A.B. Robinson, K. Irving, M. McCrea, Proc. Natl. Acad. Sci. U.S.A. 70 Ž1973. 2122–2133. w11x J.M.C. Gutteridge, S. Wilkins, Biochim. Biophys. Acta 759 Ž1983. 38–41.
w12x E. Shinar, T. Navok, M. Chevion, J. Biol. Chem. 258 Ž1983. 14778–14783. w13x L. Fucci, C.N. Oliver, M.J. Coon, E.R. Stadtman, Proc. Natl. Acad. Sci. U.S.A. 80 Ž1983. 1521–1525. w14x J.J. Gumicio, Hepatology 9 Ž1989. 154. w15x R. Gebhardt, D. Mecke, EMBO J. 2 Ž1983. 567–570. w16x R. Gebhardt, D. Mecke, in: D. Haussinger, H. Sies ŽEds.., Glutamine Metabolism in Mammalian Tissues, Springer, Heidelberg, 1984, pp. 98–121 w17x R.L. Levine, J. Biol. Chem. 258 Ž1983. 11828–11833. w18x K. Nakamura, E.R. Stadtman, Proc. Natl. Acad. Sci. U.S.A. 81 Ž1984. 2011–2015. w19x F. DaFonseca-Wollheim, Z. Klin. Chem. Klin. Biochem. 11 Ž1973. 421–426. w20x R.L. Levine, D. Garland, C.N. Oliver, A. Amici, I. Ciment, A.G. Lenz, B.W. Ahn, S. Shattiel, E.R. Stadtman, Methods Enzymol. 186 Ž1990. 464–478. w21x L.A. Jones, J.C. Holmes, R.B. Seligman, Anal. Biochem. 28 Ž1956. 191–198. w22x W.B. Rowe, R.A. Ronzio, V.P. Wellner, A. Meister, in: H. Tabor, C.W. Tabor ŽEds.., Methods Enzymol., Academic Press, New York, 17A Ž1970. 900–910 w23x H. Ohkawa, N. Ohishi, K. Yagi, Anal. Biochem. 95 Ž1979. 351–358. w24x D.H. Lowry, N.J. Rosebrough, A.L. Farr, R.J. Randall, J. Biol. Chem. 193 Ž1951. 265–275. w25x H.U. Bergmeyer, E. Bernt, Varley’s Practical Clinical Biochemistry, 5th ed., vol. 1, William Heinemann, London, 1980, pp. 741–742 w26x Douglas G. Altman, Practical Statistics for Medical Research, 1st ed., Chapman & Hall, London, 1991, p. 278 w27x R.O. Recknagel, A.K. Ghoshal, Lab. Invest. 15 Ž1966. 132–146. w28x P.E. Starke-Reed, C.N. Oliver, Arch. Biochem. Biophys. 275 Ž1989. 559. w29x K. Jungermann, D. Sasse, Trends Biochem. Sci. 3 Ž1978. 198–202. w30x R. Gebhardt, H.J. Burger, H. Heini, K.L. Schneiber, D. Mecke, Hepatology 8 Ž1988. 822–830. w31x R. Gebhardt, J. Reichen, Hepatology 20 Ž1994. 684–691.
Does oxidative protein damage play a role in the pathogenesis of carbon tetrachloride-induced liver injury in the rat? Premila N. Sundari a
a,)
, Wilfred G a , Banumathi Ramakrishna
b
Department of Biochemistry, Christian Medical College & Hospital, Vellore 632002, Tamilnadu, India Department of Pathology, Christian Medical College & Hospital, Vellore 632002, Tamilnadu, India
b
Received 28 July 1997; accepted 13 August 1997
Abstract Free radicals have been implicated in the pathogenesis of alcohol-induced liver injury in humans and carbon tetrachloride ŽCCl 4 .-induced liver injury in rats. The most extensively studied aspect of free radical induced liver injury is lipid peroxidation. Recently it has been found that free radicals can cause oxidative damage to cellular proteins and alter cellular function. One such susceptible protein is the enzyme glutamine synthase ŽGS.. The chemical effects of CCl 4 on cell proteins and their biological consequences are not known. Hence, in our study, the effect of CCl 4 on liver protein oxidation and GS activity were investigated and compared with lipid peroxidation. A significant increase in liver protein carbonyl content Ž2–3 fold. and a significant decrease in hepatic GS activity Ž44–57%. were observed. Damage to proteins was rapid in onset and increased with time. Acute exposure of rats to CCl 4 resulted in an increase in hepatic protein carbonyl content and a decrease in hepatic GS within 1 h. In cirrhosis of the liver induced by CCl 4 , the decrease in hepatic GS activity was accompanied by a significant increase in plasma ammonia levels. We conclude that protein oxidation may play a role in the pathogenesis of CCl 4 induced liver injury and that the accumulation of oxidised proteins may be an early indication of CCl 4 induced liver damage. q 1997 Elsevier Science B.V. Keywords: Carbon tetrachloride; Liver injury; Protein oxidation; Glutamine synthase; Lipid peroxidation
1. Introduction Cirrhosis of the liver is one of the major health problems in developed countries. Cirrhosis of the liver is the fourth leading cause of death in the American adults w1x, about 70% of which is associAbbreviations: CCl 4 , carbon tetrachloride; GS, glutamine synthase; PCo, protein carbonyl; LPo, lipid peroxide; MDA, malondialdehyde; TCA, trichloroacetic acid; DNPH, 2,4-dinitrophenylhydrazine; HEPES, 4-Ž2-hydroxyethyl. 1-piperazine ethane sulphonic acid; PV, perivenular; AST, aspartate aminotransferase; ALT, alanine aminotransferase ) Corresponding author. Tel.: 91-416-22603-4267; fax: 0091416-32788; E-mail: [email protected]
ated with alcoholism. Although extensive work is being done in the field of cirrhosis, the molecular mechanisms leading to cell death and increased collagen accumulation are still not clear. Therefore, treatment and prevention of the fibrotic process is still elusive. The most widely accepted animal model of cirrhosis is the ratrcarbon tetrachloride ŽCCl 4 . model, which is remarkably similar to human alcoholic cirrhosis both histologically w2,3x and systemically w4,5x. The involvement of free radicals is reported in both cases. So far, the most extensively investigated mechanism of free radical induced liver injury is lipid peroxidation w6–9x. Only recently, the possibility that free radicals may cause potential damage to proteins
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has been taken into account and a wide range of changes have been reported w10–12x. The formation of carbonyl derivatives of proteins is suggested to be a useful measure of oxidative damage to proteins. Key metabolic enzymes have been shown to be inactivated by oxygen free radicals produced by either mixed function oxidase system or by non-enzymatic methods w13x. The oxidative inactivation of enzymes by free radicals and the intracellular accumulation of oxidised proteins may play a critical role in the alteration of cellular function and cell death. The chemical effects of CCl 4 on cell proteins and the biological consequences of such reactions have not been studied. CCl 4 is metabolically activated to free radicals mainly in perivenular ŽPV. hepatocytes which are rich in the drug metabolising microsomal cytochrome P450 system w14x. GS which plays a pivotal role in nitrogen metabolism, including detoxification of ammonia is found exclusively in the PV hepatocytes. It is in fact considered to be a PV marker enzyme w15,16x. Exposure of GS to non-enzymatic mixed function oxidase system composed of ascorbate, oxygen and FeŽIII. has been shown to result in the oxidative modification of the enzyme. Oxidative modification of GS abolishes its catalytic activity w17,18x. Due to the co-localization of cytochrome P450 and GS on the microsomes of PV hepatocytes, it is likely that free radicals generated from CCl 4 by cytochrome P450 may cause oxidative inactivation of GS. The objectives of the present study were to find out Ž 1. whether oxidation of liver proteins occurs in acute and chronic CCl 4 administration; Ž2. whether there is any alteration in hepatic GS activity; and Ž 3. to find out whether there is any relationship between hepatic GS and plasma ammonia levels. Our results provide evidence for liver protein oxidation and also for a link between liver protein carbonyl ŽPCo. content and the activity of hepatic GS.
vanised iron cages and maintained on standard laboratory rat chow Žsupplied by Lipton India. with free access to tap water in a thermostatically controlled room Ž288C. under 12 h darkrlight cycle.
2. Materials and methods
2.3.2. Acute exposure to CCl 4 The rats were administered phenobarbitone in drinking water Ž500 mgrl. for 10 days. The rats were then exposed to CCl 4 vapours once for a period of 10 min as described above. The rats were sacrificed at
2.1. Animals Adult male Wistar rats weighing 150–200 g were used for the study. The rats were housed in gal-
2.2. Chemicals 2,4-Dinitrophenyl hydrazine, protease inhibitors Žleupeptin, pepstatin A, aprotinin and phenylmethylsulphonyl fluoride., HEPES buffer, ATP, g-glutamyl hydroxamate, 1,1,3,3-tetramethoxy propane and glutamate dehydrogenase were obtained from Sigma, St. Louis, MO, USA. All other reagents were of the highest purity available. 2.3. Animal treatment 2.3.1. Induction of cirrhosis Cirrhosis was induced in the rats by chronic exposure to CCl 4 vapours according to the method of McLean et al. w5x. A wooden box, with a glass front Ž72 = 45 = 46 cm, i.e., 150 l capacity. was fitted with an inlet tap and housed in a fume cupboard. Compressed air was passed via a flow meter, at 4 lrmin, bubbling through a train of two wash bottles containing ‘‘Analar’’ CCl 4 maintained at 208C, and into the box. Air left the box by leaking through the joints. CCl 4 was run in for 5 min and was then turned off and the rats left in the box for 5 more minutes Žtherefore, total period of 10 min.. Treatment was carried out twice a week for 12 consecutive weeks. Tap water containing phenobarbitone Ž500 mgrl. was the only source of drinking water for the rats 10 days prior to the first dose of CCl 4 and throughout the treatment period. At the end of 12 weeks, the rats were sacrificed. Slices of liver tissue were fixed in 10% formalin and processed and stained with haematoxylin–eosin, Foot’s reticulin and Van Gieson stains for histological assessment. The time period of 12 weeks was found to be sufficient to induce cirrhosis in the rats. Control rats were kept under the same conditions as the experimental rats, but were administered phenobarbitone alone.
34.00 " 8.81 Žns . Ž n s 6 . 72.8 " 5.76 Žns . Ž n s 5 . 23.17 " 5.08 Žns . Ž n s 6 . 65.25 " 4.57 Žns . Ž n s 4 . 20.67 " 3.27 Ž n s 6 . 65.67 " 6.86 Ž n s 6 .
Data represent mean value " SD. a P - 0.001; b P - 0.01; c P - 0.05. ns – statistically not significant compared with zero time, nd – not determined, n s no. of rats. Rats were sacrificed at various time intervals after exposure to CCl 4 vapours for 10 min. Liver was excised, homogenised and assays carried out as described in Section 2. Protein carbonyl content was calculated from extinction co-efficient of 21.0 mMy1 cmy1 for aliphatic hydrazones. One unit of GS activity is defined as the amount of enzyme that catalyses the formation of 1 m mol of gamma glutamyl hydroxamate per hour at 378C.
nd
68.75 " 19.56 Ž n s 4 . 64.25 " 10.87 c Ž n s 4 . 94.5 " 10.38 Žns . Ž n s 4 . 66.50 " 5.2 Žns. Ž n s 4 . 121.2 " 25.41 Ž n s 5 . 183.2 " 24.44 c Ž n s 5 . 85.25 " 29.32 Ž n s 4 . 89.00 " 20.94 Žns . Ž n s 4 .
c
98.57 " 6.9 Žns . Ž n s 8 .
c
nd nd nd 92.95 " 6.5 Ž n s 6 .
0.54 " 0.08 a Ž n s 6 . 0.60 " 0.04 b Ž n s 5 . 0.86 " 0.14 Ž n s 6 .
nd
c
0.48 " 0.04 a Ž n s 6 . nd 0.47 " 0.09 a Ž n s 5 .
393.6 " 132.8 b Ž n s 5 . 267.7 " 48.8 Žns. Ž n s 5 . 252.8 " 82.6 Ž n s 6 .
0.49 " 0.09 a Ž n s 5 .
822.1 " 207.3 a Ž n s 4 . 1139.6 " 192.5 a Ž n s 4 . 1877.1 " 75.6 a Ž n s 4 .
18 7.76 " 0.41 a Ž n s 3 .
14 8
nd 7.68 " 0.83 a Ž n s 6 .
4
6.53 " 0.36 a Ž n s 6 .
2
5.05 " 0.28 Ž n s 6 . 0
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Protein carbonyl content Žnmolrmg protein . Lipid peroxide level Žnmolrg wet weight. Glutamine synthase activity ŽUrmg protein . Plasma ammonia level Ž m g% . Plasma ALT ŽUrl. Plasma AST ŽUrl.
Time Žh . after a single dose of CCl 4
2.4.2. LiÕer protein carbonyl content About 250 mg of liver was minced and homogenised in 10 ml of ice-cold 10 mmolrl HEPES buffer, pH 7.4 containing 125 mmolrl KCl, 5 mmolrl Na 2 EDTA and protease inhibitors wleupeptin Ž0.5 mgrl. , pepstatin Ž 0.7 mgrl. , aprotinin Ž 0.5 mgrl. and phenylmethylsulphonyl fluoride Ž40 mgrl.x to inhibit protease activities. PCo content was measured as described by Levine et al. w20x. Aliquots of homogenate were precipitated with 20% trichloroacetic acid ŽTCA. . The pellet was suspended in 10 mmolrl DNPH in 2 molrl HCl and allowed to stand at room temperature for 1 h, with vortexing every 10 min. Proteins were then reprecipitated with 20% TCA and centrifuged. The pellet was washed thrice with 1 ml of ethanolrethyl acetate Ž1 : 1 vrv.. The final pellet was dissolved in 1 ml of 6 molrl guanidine hydrochloride Žin 20 mmolrl potassium phosphate buffer pH 2.3.. The difference in absorbance of the sample treated with DNPH in HCl was determined versus the sample treated with HCl alone at 366 nm. Results are expressed as nmoles of DNPH incorporatedrmg of
Parameter
2.4.1. Plasma ammonia Plasma ammonia was estimated enzymatically using glutamate dehydrogenase and the change in absorbance of NADPH was monitored spectrophotometrically at 340 nm w19x.
Table 1 Effect of a single dose of CCl 4 on hepatic protein carbonyl content, lipid peroxide level, glutamine synthase activity, plasma ammonia level and transaminases
The CCl 4 treated and the control rats were fasted overnight before sacrifice. Blood was collected from the animals under light ether anaesthesia by heart puncture into tubes containing Na 2 EDTA Ž 1 mgrml. and kept in ice. Plasma was separated immediately and used for the assay of ammonia and transaminases ŽALT, AST. . The livers were excised quickly, weighed and used for biochemical and histological studies. All the assays were carried out within 48 h of sacrifice of the animals.
8.66 " 0.95 a Ž n s 6 .
2.4. Biochemical assays
394.7 " 141.8 c Ž n s 6 .
28
the end of various time intervals Ž2, 4, 8, 14, 18 and 28 h.. In another experiment, the rats were exposed to a low dose of CCl 4 Žfor 6 min instead of 10 min. and sacrificed after 1 h. Phenobarbitone treated rats served as controls.
9.61 " 0.28 a Ž n s 4 .
P.N. Sundari et al.r Biochimica et Biophysica Acta 1362 (1997) 169–176
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Fig. 1. Correlation between hepatic PCo content and specific activity of GS. The regression equation is y s 1.324 y 0.0929 x Ž r s y0.847, P - 0.001..
Fig. 2. Time dependent changes in PCo content, LPo level and GS activity in rat liver after a single dose CCl 4 .
protein calculated from an extinction coefficient of 21 mMy1 cmy1 for aliphatic hydrazones w21x.
2.4.5. Plasma transaminases Plasma aspartate aminotransferase Ž AST. and alanine aminotransferase ŽALT. activities were assayed as described by Bergmeyer and Bernt w25x.
2.4.3. Hepatic glutamine synthase actiÕity About 1 g of liver was minced and homogenised in 10 ml of ice-cold HEPES buffer pH 7.4 containing 125 mmolrl KCl and 5 mmolrl Na 2 EDTA. The homogenate was sonicated for 30 s at 200 V ŽVibronics, India.. The homogenate was centrifuged for 15 min at 16,000 = g at 48C and the supernatant was used for the determination of GS activity and protein. GS activity was assayed by the method described by Rowe et al. w22x. g-glutamyl hydroxamate was used as the standard. One unit of enzyme activity is defined as the amount of enzyme catalysing the formation of 1 mmol of g-glutamyl hydroxamate per hour at 378C.
3. Statistical analysis All parameters are expressed as mean values " S.D. Differences between mean values of tests and controls were evaluated statistically by Student’s t-test. A P value of - 0.05 was regarded as
Table 2 Hepatic protein carbonyl content, glutamine synthase activity and plasma transaminase levels 1 h after exposure of rats to low dose of CCl 4 vapours Parameter
2.4.4. Hepatic lipid peroxide leÕels One gram of liver was homogenised in 9 ml of 1.15% ice-cold KCl. Lipid peroxide ŽLPo. levels were measured in whole homogenate by the thiobarbituric acid reaction w23x. Results are expressed as nmoles of malondialdehyde Ž MDA. per gram wet weight of liver. Protein was measured by the method described by Lowry et al. using bovine serum albumin as standard w24x.
Hepatic PCo content Žnmolrmg protein. Hepatic GS activity ŽUrmg protein. Plasma ALT ŽUrl. Plasma AST ŽUrl.
Control rats Ž ns6.
CCl 4 treated rats Ž ns 7.
5.10 " 0.16
6.22 " 0.44 a
0.88 " 0.11
0.72 " 0.10 b
19.71 " 4.54 68.17 " 8.42
20.43 " 4.72 Žns. 70.57 " 7.66 Žns.
n s no. of rats. Data represent mean value " SD. P - 0.001; b P - 0.05. Rats were sacrificed 1 h after exposure to CCl 4 vapours for 6 min and assays carried out as described in Section 2. a
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Fig. 3. Rat liver showing normal appearance and no necrosis 1 h after exposure to low dose of CCl 4 vapours. PT – portal tract, ThV – terminal hepatic vein. The stain used was hematoxylin–eosin Ž=90..
Fig. 4. Liver cirrhosis in rats treated with CCl 4 for 12 weeks. The stain used was Foot’s reticulin Ž=40..
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Table 3 Hepatic protein carbonyl content, lipid peroxide level, glutamine synthase activity and plasma ammonia level in CCl 4-induced cirrhosis of the liver Parameter
Control
Cirrhosis
Protein carbonyl content Žnmolrmg protein. Lipid peroxide level Žnmolrg wet weight. GS activity ŽUrmg protein. Plasma ammonia level Žmgr100 ml.
4.12 " 0.39 Ž n s 6. 379.3 " 36.5 Ž n s 7. 1.00 " 0.21 Ž n s 8. 122.6 " 26.3 Ž n s 6.
11.19 " 1.02 a 551.3 " 92.5 b 0.43 " 0.10 a 262.5 " 77.6 b
Ž n s 8. Ž n s 8. Ž n s 10. Ž n s 10.
n s no. of rats. Data represent mean value " SD. P - 0.001; b P - 0.01. Rats were sacrificed after 12 weeks of treatment with CCl 4 as described in Section 2.
a
statistically significant. Karl Pearson’s test w26x was used for correlation studies.
4. Results 4.1. Acute exposure to CCl 4 Table 1 shows the time dependent effect of single dose of CCl 4 on hepatic PCo content, LPo level, GS activity, plasma ammonia level and transaminase levels. A significant increase in PCo content was observed Žapproximately 2 fold-28 h value. . This was accompanied by a significant decrease in GS activity Ž44%.. A clear significant inverse correlation be-
tween PCo content and GS activity was observed as shown in Fig. 1 Ž r s y0.847, P - 0.001. . A significant increase in hepatic MDA levels was also observed Ž7 fold-14 h value. . Fig. 2 shows time dependant changes in the above mentioned parameters. Liver PCo began to rise significantly as early as 2 h and showed an increase with time. Coincidentally, hepatic GS activity also began to fall at 2 h and remained low for 28 h. MDA level showed significant elevation at 4 h, peaking at 14 h and declining thereafter. There was no significant difference in plasma ammonia levels between the CCl 4 treated rats and the untreated rats. Plasma transaminases began to rise at 8 h, peaked at 14 h and began to decrease thereafter suggesting that hepatic necrosis had occurred to a certain extent in the later hours. In the rats sacrificed 1 h after the low dose of CCl 4 , the hepatic PCo level was increased by 18% and the activity of GS was decreased by 18% as shown in Table 2. There was no evidence of necrosis histologically as shown in Fig. 3. Plasma ALT and AST were unaltered. 4.2. Chronic liÕer injury
Fig. 5. Relationship between the specific activity of hepatic GS and plasma ammonia concentration in cirrhotic rats. The regression equation y s 522.815 y 661.997x Ž r s y0.819, P 0.05..
The rats that were exposed to CCl 4 vapours for 12 weeks developed cirrhosis as shown in Fig. 4. Table 3 shows the effect of chronic exposure of rats to CCl 4 on liver PCo, LPo, GS and plasma ammonia level. Nearly 3 fold increase in PCo was observed. GS activity in the cirrhotic rats was decreased significantly Ž57%.. A moderate, but significant increase in MDA levels Ž1.5 fold. was also observed in the cirrhotic rats. Plasma ammonia levels were elevated more than 2 fold in the cirrhotic animals and they
P.N. Sundari et al.r Biochimica et Biophysica Acta 1362 (1997) 169–176
showed significant inverse correlation with hepatic GS activity as shown in Fig. 5 Ž r s y0.819, P 0.05..
5. Discussion It is a well established fact that lipid peroxidation plays a major role in CCl 4 induced development of fatty liver and cirrhosis. Accordingly, we found significant increase in the MDA levels Ž7 fold-14 h value. upon single exposure to CCl 4 vapours. The decrease in MDA levels after 14 h may be due to the metabolism of MDA by the rat liver mitochondria w27x. A significant increase in the PCo content of the liver Ž approximately 2 fold-28 h value. was also observed. Nearly 2 fold increase in liver PCo after single dose of CCl 4 may not appear significant. However, it has been estimated that values as low as 2 nmol of carbonyl per mg protein represents damage to about 10% of total cellular proteins w28x Žthese are minimal values as the oxidative modification of some amino acid residues in protein does not lead to the formation of carbonyl derivatives. . Extrapolating from this, we could calculate that, in CCl 4 treated rats about 23% of total hepatocellular proteins are damaged. As mentioned earlier, we found significant increase in PCo levels even within an hour of exposure to CCl 4 vapours. This suggests that the accumulation of oxidised proteins in the liver may be an early indication of CCl 4 induced liver damage. Decrease in hepatic GS activity was observed upon single exposure to CCl 4 vapours. GS is present exclusively in the PV hepatocytes and is highly susceptible to oxidative damage by free radicals. The decrease in GS activity in acute CCl 4 administration may be due to the necrosis of PV hepatocytes or due to the oxidative inactivation of the enzyme. An increase in PCo content and a decrease in GS of the liver in the absence of necrosis would suggest oxidative inactivation of the enzyme. We found a significant increase in PCo levels and a significant decrease in GS in the liver 1 h after exposure to CCl 4 vapours Ž Table 2. , when there was no evidence of hepatocellular necrosis both histologically and biochemically. Therefore, decreased GS activity in acute CCl 4 administration can be attributed to the oxidative inac-
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tivation of the enzyme by trichloromethyl radical and trichloromethylperoxy radical generated from CCl 4 . Although significant decrease in hepatic GS was observed in acute exposure to CCl 4 vapours, plasma ammonia levels were unaltered. This could be explained on the basis of the fact that the other important enzymes concerned with the detoxification of ammonia namely the urea cycle enzymes are mainly periportal in their location w29x and are reported to be unaffected by CCl 4 , the PV toxin in short term exposure of rats w30x. Regarding chronic liver injury, a greater increase in liver PCo was observed Ž approximately 3 fold. compared to acute liver injury Ž approximately 2 fold.. As mentioned earlier values as low as 2 nmolrmg protein represents damage to about 10% of total cellular proteins. Extrapolating from this in CCl 4 treated rats about 31% of total hepatocellular proteins are damaged. A 1.5 fold increase in MDA levels was also observed. Such a low value in MDA levels may be due to the metabolism of MDA by the rat liver mitochondria as mentioned earlier. In chronic liver injury, the decrease in hepatic GS can be attributed mainly to the loss of functional hepatocytes. The significant inverse correlation between plasma ammonia levels and hepatic GS in the cirrhotic rats suggests that the decrease in the activity of hepatic GS may contribute to hyperammonemia observed in the cirrhotic animals, while other factors such as portosystemic shunting play important roles. A similar observation has been made earlier by Gebhardt and Reichen w31x, although they found a greater decrease in GS activity Ž80%. compared to our study Ž57% reduction.. These authors have also suggested that the decrease in GS activity in cirrhosis of the liver contributes to hyperammonemia. Our study suggests that oxidative damage to proteins occurs in acute as well as chronic exposure of rats to CCl 4 and that it may contribute to the pathogenesis of liver injury, while lipid peroxidation plays a major role. Our results also suggest that the accumulation of oxidised proteins in the liver may be an early indication of CCl 4 liver injury and that the decrease in hepatic GS activity Ž in the absence of hepatocellular necrosis. may be due to the oxidative inactivation of the enzyme. The decrease in GS activity in chronic liver injury induced by CCl 4 can be attributed to the loss of functional hepatocytes. The
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decrease in GS may contribute to hyperammonemia observed in cirrhotic animals, while factors such as portosystemic shunting play important roles. Acknowledgements This work was supported by the grants from the FLUID Research Fund, Christian Medical College & Hospital, Vellore-4. We are grateful to Mr. Tamilselvan for his technical assistance. References w1x S. Sherlock, J. Dooley, Diseases of the Liver and Biliary System, 9th ed., Blackwell Scientific Publications, Oxford, 1993, p. 370 w2x R.J. Stenger, Arch. Pathol. 81 Ž1966. 439–447. w3x E. Rubin, H. Popper, Medicine 46 Ž1967. 163–183. w4x P.M. Daniel, M.M.L. Prichard, P.C. Reynell, J. Pathol. Bacteriol., 64 Ž1952a. 53–60 w5x E.K. McLean, A.E.M. McLean, P.M. Sutton, Br. J. Exp. Pathol. 50 Ž1969. 502–506. w6x E. Albano, A. Tomasi, L. Goria-Gatti, M.U. Dianzani, in: C. Rice Evans ŽEd.., Free Radicals, Cell Damage and Disease, Richelieu Press, London, 1986, pp. 117–126 w7x E. Albano, A. Tomasi, L. Goria-Gatti, M.U. Dianzani, Chem. Biol. Interact. 65 Ž1988. 223–234. w8x E. Albano, A. Tomasi, J.O. Persson et al., Biochem. Pharmacol. 41 Ž1991. 1895-1902 w9x I. Johansson, G. Ekstrom, B. Scholte, D. Puzycky, H. Jornall, M. Ingelman-Sundberg, Biochemistry 27 Ž1988. 1925–1934. w10x A.B. Robinson, K. Irving, M. McCrea, Proc. Natl. Acad. Sci. U.S.A. 70 Ž1973. 2122–2133. w11x J.M.C. Gutteridge, S. Wilkins, Biochim. Biophys. Acta 759 Ž1983. 38–41.
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