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November 1988, Volume 38, Issue 11

Original Article

POTENTIATING EFFECTS OF PHENOBARBITONE ON THE INDUCTION OF CIRRHOSIS IN RATS BY CARBON TETRACHLORIDE

Fazli M. Qazi  ( Department of Pathology, Basic Medical Sciences Institute, Jinnah Postgraduate Medical Centre, University of Karachi, Pakistan. )
S. Mahmood Alam  ( Fazli M. Qazi Department of Pathology, Basic Medical Sciences Institute, Jinnah Postgraduate Medical Centre, University of Karachi, Pakistan. )

Abstract

The potentiating effects of phenobarbitone sodium on the experimental induction of liver necrosis and cirrhosis by carbon tetrachloride (CC14) were studied in 50 rats.
CC4 was given by intraperitoneal injection, phenobarbitone in drinking water. Thirty rats received both agents; and 10 each received only one agent. In 7 of 10 animals given CCI4 alone, moderate cirrhosis without ascites developed within 6 to 8 weeks; the other 3 rats died before fourth week. All animals receiv­ing both agents and surviving for as long as 5 to 8 weeks developed severe cirrhosis with marked proli­feration of the bile ducts within 2 months. Most of these animals also had ascites. Five rats given only phenobarbitone remained healthy, although their livers increased in weight (JPMA 38: 1988).

INTRODUCTION

Liver cirrhosis in rats can be produced by dosing them twice weekly with carbon tetrachlo­ride (CCl4) 1-3 but this method has some draw­backs. A long time (up tc 20 weeks in some instances) is required to produce cirrhosis, and many animals die intercurrently. In some animals cirrhosis does not develop even after prolonged dosage4
Acute toxicity may develop when CC14 is metabolized by microsomal-hydroxylating enzyme systems of the liver5. The toxicity is augmented by agents that activate this enzyme system - e.g., phenobarbitone sodium and DDT6-7 In stock rats, the LD50 for CCL is about 5 mL/kg body weight, while in rats treated with phenobarbitone, the LD50 is only 0.5 mL/kg4.
To further study the potentiating effect of phenobarbitone on the induction of cirrhosis by CCI4, an experimental study was designed in which twice-weekly doses of carbon CC14 were administered by intraperitoneal (i.p.) injection. Other workers have given the drug by stomach tube, subcutaneous injection or inhalation. Pheno­barbitone was given by mixing in drinking water.

MATERIAL AND METHODS

Animals
Fifty female albino rats weighing 200 to 300 g were maintained on Gram soaked in water for 24 hours and then sprinkled with milk powder. The animals were divided into 6 groups (Table)~TABLE. Treatment Protocol for the Six Gnups of Rats Chemical Agents
CC14, 0.5 mI/kg, was given by intraperi­tonealy twice weekly for 8 weeks to 40 rats in groups B, C, D and E. One week before the first injection, water-soluble phenobarbitone sodium (E. Merck), in amounts ranging from 0.125 to 0.5 g/L, was added to the drinking water supplied to the rats in groups C, D, E and F for period of 9 weeks. Five rats in group A served as controls, receiving twice-weekly i.p. injections of distilled water, 0.5 mL/kg. Group F (n = 5) received, in addition to the i.p. injections of distilled water, the maximum dose of phenobarbitone sodium (0.5 g/L) in their drinking water (Table).


Autopsy
Autopsies were performed on all animals. After gross examination of the viscera, any ascitic fluid present was collected in sterile tubes for bacterial culture, routine cytological studies and determination of protein content.
Histologic Examination
Ten percent solutions of buffered formalin and 10% alcoholic formalin were used as fixatives. Sections of liver tissue were stained with hema­toxylin and eosin, Gomori’s reticulin, trichrome, PAS, Best’s carmine, methyl green thionine and oil red 0. stains.

RESULTS

The animals in Group A remained healthy, voided urine of normal color, and gained weight, the average gain being 8.7% of their initial body weight. At autopsy, the average weight of their livers and spleens was 6.4g and 0.4g respectively.
All the animals in group B became sluggish, looked sick and voided yellow urine. Mean weight loss was 12% of their initial body weight. Three animals (30%) died within 2 weeks. Autopsy showed swollen livers, covered by yellowish spots, more marked in the smaller lobes. Micros­copy showed acute massive centrilobular necrosis in 2 and necrosis with lobular collapse in 1 rat.
Of 7 animals (70%) that survived till 7 weeks, autopsy showed finely nodular livers. Microscopic examination of hepatic sections revealed cirrhosis of the micronodular type. Their livers and spleens were enlarged, weighing on the average 9 g and 1.1 g, respectively.
The rats in group C appeared sicker than those in group B. Their urine was yellow, and they lost an average of 14.7% of their initial body weight. Six animals (60%) died in the first week and had swollen, dark-red livers, some of which had black margins. Sections showed massive centri­lobular necrosis. In the 4 animals (40%) that survived the experimental period, autopsy revealed micronodular cirrhosis in 3 and mixed nodular cirrhosis in 1. All 4. animals had marked proli­feration of the bile ducts and widespread fatty change. Measurable ascites was present in 3 of 4 rats. Their livers and spleens were enlarged, weighing on the average 10.4 g and 12 g.
In group D, the average weight loss was 13.3%. All animals looked sick, and 4 (40%) died within 2 weeks. Gross fmdings in these were similar to those in group B, while the histo­logical picture was similar to that seen following the early deaths in group C.
Six animals in group D (60%) survived 8 to 9 weeks. In all of these, measurable ascites was present, and microscopic examination of hepatic sections revealed micronodular cirrhosis with fatty change (Figure 1).

Their liver and spleens were enlarged, weighing on the average 9.25 g and 1.4 g, respectively.
In group E, the general observations were similar to those in groups C and D the average weight loss being 13.3%. Six animals (60%) died in the first 3 weeks, and at autopsy showed gross and microscopic changes (Figure 2)

comparable to those observed following early deaths in groups C and D. Of 4 animals that survived to the end of the experiment, 3 had micronodular cirrhosis (Figure 3)

and 1 had mixed nodular cirrhosis (Figure 4). Ascites was present in 2. As in groups B, C and D, the livers showed fatty changes. Their livers and spleens were enlarged, weighing 10.1 g and I .4g on the average.
All 5 animals in group F looked healthy, but their livers were enlarged, weighing an average of 112 g as compared to 6.4 g in the control animals.
Examination of Ascitic Fluid
Cultures did not yield any bacteria. Cyto- logic studies showed lymphocytes, and mesothelial cells only. The protein contein content ranged form 1.8 g/100 mL to 3 g/100 mL. These findings were similar in all the cirrhotic animals of groups C,D, and E with ascites.

DISCUSSION

For at least half a century, it has been known that chloroform, Cd4 and phosphorous are hepatotoxic, and that cirrhosis of the liver can be produced in rats by the administration of CCl1-3 (Forbes, 1939).
In the present study, a high percentage of animals receiving CCl4 alone (group B) survived the experimental period and developed definite cirrhosis of the micronodular type. The fact that this yield of cirrhosis is higher than those recorded by others3-4 may be explained by one or more of the following factors:
1. Animals in the present study might have been more susceptible to the hepatotoxic action of CC!4. Host factors are always important in the pathogenesis of liver necrosis and cirrhosis. Among alcoholics, for example, cirrhosis develops in only 10% to 30%8
2. Present animals might have suffered from deficiency of certain dielary factors e.g., protein or  vitamins - which have a protective effect on the liver.
3. No glycogen was found in the livers of present animals (when they were subjected to PAS and Best’s carmine staining), although all liver sections from the control group showed some traces of glycogen. The lack of glycogen — possi­bly caused by a deficiency of carbohydrate in the diet —  might have rendered these animals more vulnerable to hepatic injury by CCI4 and conse­quent cirrhosis.
Although none of the animals in group B showed ascites, the omentum appeared edema­tous to the naked eye. McLean et at4 reported a similar observation.
The potentiating effect of phenobarbitone on the hepatic injury induced by CC!4 was demon­strated in groups C, D and E. Acute CC14 toxicity with massive centriobular necrosis led to the early death of 533% of the subjects in these 3 groups. Most of the animals surviving for 7 to 8 weeks in groups C,D and E (40%, 60%, 40% respectively) showed severe micronodular cirrhosis at autopsy. Only 2 animals (1 in group C and 1 in group E) had mixed nodular cirrhosis. The severity of the cirrhosis in these 3 groups was shown by the total loss of normal lobular architecture, by thick fibrous-tissue bands delineating parenchyma! pseudonodules, and by marked proliferation of bile ducts. These findings were less marked in group B (the rats receiving CCl4 alone).
The presence of measurable ascites in 75%, 100% and 50% of the cirrhotic animals in groups C, D and E is another measure of severity of cir­rhosis in these groups as compared to group B. McLean et aI4 reported ascites in 50% of their cirrhotic animals.
The increased liver weight among group F animals is consistent with other reports4-9-10  Argyris (1968), in phenobarbitone-treated rats. Histological study of the livers from group-F rats showed normal lobular architecture but many binucleated cells.
The spleens of cirrhotic animals weighed 1 .lg to 1 .4g as compared to 0.4g in the controls. McLean et al4 also reported increased spleen weight in their cirrhotic animals (1-2 g as compared to 0.7 g in their control group).

ACKNOWLEDGEMENT

We are indebted to Professor N. A. Jafarey, Professor of Pathology, Basic Medical Sciences Institute, Jirinah Postgraduate Medical Center, Karachi, for his valuable suggestions and guidance during the study. We also wish to acknowledge the expert assistance of Mrs. Patt Moore, Depart­rnent of Pathology, The Bowman Gray School of Medicine, Winston-Salem, NC, USA, in manus­cript preparation.

REFERENCES

1. Cameron, G.R. and Karunaratne, W.A.E. Carbon tetrachioride cirrhosis in relation to liver regene ration. J. PathoL Bacterial., 1936; 42:1.
2. Forbes, J.C. Studies on the prevention of liver cirrhosis by the subcutaneous injections of    xantlune   containing preparations. 3. Pharmacol. Exp. Ther., 1939; 65: 287.
3. Oliver, R.H.P. and Sutton, P.M. The effects of partial hepatectomy on portal pressure in rats. Br. j   Surg., 1966; 53: 138.
4. McLean, E.K., McLean, A.E.M. and Sutton, P.M. Instant cirrhosis; an improved method for producing cirrhosis of the liver in rats by simultaneous administration of carbon tetrach­loride and phenobarbitone. Br. J. Exp. Pathol., 1969; 50:5 02.
5. Garner, R.C. and McLean, E.M. Increased susce­ptibility to carbon tetrachboride  poisoning in the rat after pretreatment with oral phenobarbitone. Biochem. Pharmacol., 1969; 18:645.
6. McLean, A.E.M. and McLean, E.K. The effects of diet and l,1,1-trichloro-2, 2, bis-(P.chloro­phenyl) ethane (DDT) on microsomal hydroxy­lating enzymes and on   sensitivity of rats to carbon t trachioride poisoning. Biochem. J., 1966k 100:564.
7. Marshall, WJ. and McLean, A.E. The effect of oral phenobarbitone on hepatic    microsomal cytochrome P.450 and demethylation activity in rats fed normal and low protein diets. Biochem. Pharmacol., l969~ 18 : 153.
8. Popper, H. Pathologic aspects of cirrhosis; a review. Am. j Pathol., 1977; 87:228.
9. Argyris, T.S. Liver growth associated with the induction of aminopyrine demethylase activity after phenobarbital treatment in adult male rats. J. Pharmacol. Exp. Ther., 1968; 405.

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