Exp. Path. 2H, 33-40 il ~IK~ I

Laboratoiru des Int('r<H,tions CPllnlain's, l'ninrsit6 de Bordeaux II. F - 33076 Bordeaux Cedex, Frame

Acute hepatotoxicity of carbon tetrachloride. Different liver lobes response in rats with portal branch ligation

By P. BIOULAC-SAGE, Y. LAPOSTOLLE, N. MORE and Cn. BALABAUD

With 5 figures

(Received September 16, 1983)

Address for correspondence: Prof. Dr. PArLETTE BIOUL.\C-SAGE, Laboratoire des Interactions Cellu­laires, Universite de Bordeaux II, 146, rue Leo-Saignat, F - 33076 Bordeaux Cedex, France.

Key words: hepatotoxicity, ('arbon trtrarhloride; liver, drug metabolism; portal branch ligation; hepatic blood flow

Summary

Portal branch ligation (PBL) leads to atrophy of the corresponding lobe (AL) and hypertrophy of the non ligated lobes (HL). Liver architecture of AL is preserved. In this model we assessed in rats, one month after PBL, the acute hepatotoxicity of carbon tetrachloride (CCI4) given i.p. or i.v. Grading of necrosis, steatosis and ballooning demon­strated that at 24 and 48 h, AL were less seyerely damaged than HL and lobes of sham operated rats. Changes in hepatic blood flow, drug metabolism and/or in sinusoidal cells may represent cauges invoh'ed in the partial he1Jato-protection.

Introduction

Hepatocytes along sinusoids receive mixed blood of portal and arterial origin. Morpholo­gical and functiomd tomequenees of portacaval shunt (PCS) (3, 7) or ligation of the hepatic artery (10) have been extensively studied. On the contrary, very little is known when vas­cularization disturbances affect only part of the liver without damaging the whole liver (11, 12). The division of the rat liver into several lobes offers the possibility to study such a model. The aim of this study was to investigate the acute hepatotoxicity of carbon tetra­chloride (CCI4) on tIlt' different Iobrs of the liver in rats with portal branch ligation (PBL).

111 ate rials and 111 ethods

Rat Model Female Wistar rats (180 g) were bred in our laboratory, submitted to an artificial daylight cycle,

and given free access to water and food (A04 , DAR, Villemoisson-Sur-Orge, France). Rats were given ether anesthesia, and right PIlL was performed using a surgical microscope. The right portal branch was divided between ligatures (silk 07). Care was taken to avoid arterial branches and bile duct belonging to the right lobe. Sham operated rats were used as control.

Drug administration One month later, rats (PBL and Sham PBL) received CCI4 either intraperitoneally (i.p.) or

intragastric (i.g.) as a 20% (vol/vol) solution in corn oil at a dose of 5 ml/kg. Additional rats received corn oil only. 24 h later, after an overnight fast, they were killed. In additional experiments, PBL rats injected with CCl4 (i.g) were killed 48 h and one week after the injection. Rats were anesthetized with sodium pentobarbital, bled and their liver was removed and weighed. The lobes of the liver were cut arcording to fig. 1 and weighed separately.

3 Exp. Path. 26, H. 1 33

Table 1. Linr \V('ight and bioehl'llIica\ linr function tests :!4 h ,tIter CC14 injection in rats with PBL -+

Drug n

L.wt x100 B.wt

R.Lwt x100 B.wt Bilirubin (~moljL) ALT(lUjL) AST(lUjL) Alkaline phosphatase (lUlL)

n = number of animals in each group o = unfasted rats

Sham PBL

Contro\O Olive oil CClt i.p. 4 4 4

3.95 ± 0.16+ 3.11 ± 0.19 4.18 ± 0.16*

Ull ± 0.03 0.764 ± 0.032 0.958 ± 0.049*

1.7 ± 0.5 1.7 ± 0.5 9.0 ± 10* 135 ± 40 128 ± 20 5634 ± 4692* 60 ±:!O 41 ± 8 596 ± 214*

102 ± 55 111 ± 62 111 ± 46

+ = Mean ± ISD * = P < 0.05 versus olive oil of the same experimental group (24 h fasted rats).

Fig. 1. Schematic representation of rat liver lobes. M: median; I.: left; R: right; C: caudates; I: intermediary (around the veny cava).

Light microscopic study Midsections of the left, caudate, intermediary (see fig. 1) and right lobes perpendicular to the

vascular axis were processed for light microscopy. Sections were fixed in Bonin's solution and em­bedded in paraplast and piccolyte. 3 ~m sections were stained with hematoxylin and eosin (HE). Liver histology was interpreted by one of us (P. B-S). Blind reading (i.e. the difference between the right lobe and other lobes) was impossible in rats with PBL because of the too different size and shape of the sections.

The entire slide was scanned and the pathological changes were arbitrarily scored as follows: necrosis 0 to 4+ (0, no necrosis; 1 +, focal necrosis of 1 or 2 cells per lesion; 2 +, focal necrosis of more than 2 cells per lesion; 3+, massive confluent necrosis; and 4+, zonal massive necrosis plus necrotic bridging between terminal hepatic veins) (1); steatosis 0 to 3+ (0, no steatosis; 1+, light; 2+, moderate; 3+, intense); ballooning 0 to 4+ (0, no ballooning; 1+, few ballooned cells per lesion; 2+, more than 10 ballooned cells per lesion; 3+,1 or 2 rows of ballooned cells around the necrotic zones per lesion; 4+, more than 2 rows of cells around the necrotic zone per lesion). Results were compared using the U-test of Mann and Whitney. P values < 0.05 were considered significant.

Biochemical studies Plasma bilirubin, alkaline phosphatase, alanine and aspartate aminotransferases (ALT, AST)

were measured by standard methods in use at the clinical chemistry laboratory of the University Hospital. Results were compared using the Student's t-test. P values < 0.05 were considered sig­nificant.

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PBL

CCl4 i.g. Control Olive oil CCI4 i.p. CClt i.g. 4 4 4 6 6

4.09 ± 0.20* 3.90 ± 0.16 3.03 ± 0.20 4.09 ± 0.15* 4.11 ± 0.10*

0.992 ± 0.025* 0.185 ± 0.038 0.139 ± 0.048 0.304 ± 0.070* 0.219 ± 0.066*

5.5 ± 0.6* 1.6 ± 0.4 1.75 ± 0.6 4.7 ± 0.5* 3.8 ± 1.3* 2539 ± 790* 150 ± 35 112 ± 12 1356 ± 747* 2548 ± 3530* 949 ± 434* 90± 30 58 ± 27 353± 181* 620 ± 798* 85 ± 18 130 ± 27 106 ± 54 103 ± 36 89 ± 22

Table 2. Liver histology: quantification of necrosis, steatosis and ballooning

Drug Sham PBL PBL

(n) Oil eCI4 i.p. CCl4 i.g. Oil eel4 i.p. eOl4 i.g.

4 4 4 4 6 6

Histology R L R L R L R L R L R L

Necrosis 0 4 4 0 0 0 0 4 4 0 0 0 0 1+ 0 0 0 0 0 0 0 0 1 0 6 1 2+ 0 0 0 0 1 1 0 0 5 0 0 2 3+ 0 0 0 3 2 3 0 0 0 4 0 1 4+ 0 0 4 1 1 0 0 0 0 2 0 2 pa < 0.05

Steatosis 0 4 4 0 0 0 0 4 4 3 0 2 0 1+ 0 0 0 0 0 0 0 0 3 0 3 0 2+ 0 0 :2 :2 1 1 0 0 0 1 1 0 3+ 0 0 :2 :2 3 3 0 0 0 5 0 6 pa < 0.05

Ballooning 0 4 4 0 0 0 0 4 4 5 0 2 0 1+ 0 0 4 4 3 4 0 0 1 4 2 4 2+ 0 0 0 0 1 0 0 0 0 2 2 2 3+ 0 0 0 0 0 0 0 0 0 0 0 0 4+ 0 0 0 0 0 0 0 0 0 0 0 0

n = number of rats R = right lobe; L = left lobe .. = statistical significance. Data from the right lobe and the left lobe are compared by the test

of Mann and Whitney. Data from one experimental group (sham PBL or PBL) were pooled whether 00L4 was given i.p. or i.v.

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Fig. 2. Portal braueh ligation llIo(lel Liver histology of the left lobe (hypertrophied lobe): normal appearance (PS: portal span'; SV: sllbhepatie vpin). HE; x 130.

Fig. 3. Portal branch ligation model -- Liver histology of the right lobe (atrophied lobe) in the same mumal as in fig. 2: enlarged sinusoids (PS: portal space; SV: subhepatic vein). HE; x 160.

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Fig. 4. Portal branch ligation model Liver histology of the ll'ft lobe :H h after CC1 4 injedion (i.p.): massive centrolobnlar nl'('J"osis (3 -:- ) mix('d with steatosis (:2+) (PS: portal space; SV: sub­hepatic vein). HE; x 130.

Fig. 5. Portal bmneh ligation morlel - Liver histology of th e right lobe 24 h after CCl4 injection in the same ani mal as in fig . 4: few ecntrolobular necrotic cells (1 + ), mixed with moderate steatosis (1+) (PS :portal space; SV: subhepatic vein). HE; x 160.

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Re.';u/t,

The model

One month after right PBL, the right lobe atrophied (AL) and the other lobes hyper­trophied (HL) so that liver weight/body weight ratio remained constant (table 1). Macro­scopically, there was a dear-cut color difference between the right lobe and the other lobes. The bright eolor of the right lobe looked like that observed in livers after PCS. The change in color followed a line along the right side of the vena cava. By light microscopy, there was no differenee between HI, of PBL rats and lobes of sham PBL rats. On the other side, some differences betwel'n HL (fig. 2) and AL (fig. 3) were noticed. AL architecture was slightly disorganized with sometimes enlarged sinusoids, moderate fibrosis around peri­portal spael's and el'ntrolobular veins. In addition hepatie arteries were thickened and bile ducts had a tendency to prolifera tp,

Carbon tetrachloride toxitit,v

24 h after CCl4 injection, liver weight/body weight ratio (L. wt/B. wt) and right lobe weight/body weight ratio (R. 1. wt/B. wt) increased in sham PBL and PBL rats. Hyper­trophy of the right lobe was more conspicuous in the last group. Bilirubin and aminotrans­ferases rose similarly in both groups. Results were not significantly different in each group (PBL and sham PBL) wlH'ther ('('14 was given i.p. or i.g. (table 1).

By light microBeop)". CCl4 tn:atl'd animals showed e1assirallesions ascribed to this che­mical but with obviom differenees lwtween HL and AL (table 2). In HL (fig. 4) lesions were identical to thosp obst'rwd in sham I' E1, rats with extensive necrosis (3 + to 4 +) and acute inflammation of centrolobular zone~, surroundrd by balloonrd eells and steatosis (3 +). In AL (fig. 5) lesions wrre lTlueh less ~evere, with a mild eentrolobular necrosis (1+ to 2+) and inflammatory reaction, ~ol1letimes surrounded by a few ballooned cells and moderate steatosis (0 to 2 +). In sham operatrd rats there were no histological differences between lobes.

48 h after CCI4 injrction. 1. wt/E. wt and R. L. wt/E. wt remained inc-reased (4.33 ± 0.71 and 0.23 ± 0.10 rpspe('tively) in PBL rats. Liver funetion tests remained abnormal but returned towards normal valul'~ in both groups. Histological damage was still present in HL and AL but to a somewhat lesspr degrpe (necrosis 1 to 3+, steatosis 3+ in HI,; necrosis 0 to 1 +, steatosis 1 to 2 ~ in A L).

1 week after CCl4 injection, L. wt/B. wt and R. L. wt/E. wt returned to normal (3.08 ± 0.19 and O.Hi ± 0.02 respectiHly in PBL rats) as well as liver function tests. Liver histology was almost normal in HI, with the exception of a moderate steatosis (2 to 2+). In AL, fibrosis (periportal and (,entrolobular) was more marked than in PBL rats receiving corn oil.

Discussion

The right PBL model offers the opportunity for studying the effect of portal blood flow interr.uption in the right lobe. We have cheeked in preliminary experiments that this model did not lead to portal hypertension and that there was no revascularization of the right lobe from the portal vein. Therefore this lobe is vascularized only by the hepatic artery. In this respect it resembles a portaeaval shunt (peS). Liver atrophy (11, 12), architectural dis­organization, fibrosis of portal tracts and ('entral veins, moderate proliferation of bile ducts and hyperplasia of thr hepatie artery in portal trads are ('ommon features observed in PCS (3,7,4,8). However it might not bp pntirely so because nutrients, hormones, bile salts, ek ... substances present in the portal vein art' taken up by the lobes re('eiving portal blood so that little is left to the AL. whereas in PCS these substances finally reached the liver. The present data haw e1earl~' shown that in PBL, after CCI4 administration there was a elear­eut differt'nee betwl'pn atrophic and hypertrophic lobe,: AI, was less severely damaged than HL and its weight iner!'a,€' wa~ grpater. There was no obvious damage difference between HL of PEL and lohr, of ~hall1 PEL.

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eel,. hcpato!'cllula[ death j, thp [e,ult of a sequence of events. whi<:"h ineludes (5, 13):

1" uptake of eel4 by hepatocytes 2. cytoehr()me~ P. 450 deppndent generation of triehlormethyl free radicals 3. peroxidatin' decompm,itinn of fatty adds of membrane phospholipids 4. release into eytns(,l of f'olublr t(1xie produets of lipid peroxidation 5. damage to the plaww membrane 6. increased permeability of plasma membranes to Ca2-t- ions.

Several agrnts haw been ,;]1own either to inerruse (9) or deerease (1, 2, 13) eel,. hepa­toxicity.

The mechanisms by which PBL prevents eel,. hepatotoxicity are unknown. Several factors could aet in opposite direction with a final protective effect. 1. changes in hepatic blood flow. eel,. reached hepatocytes with blood. The route of administration of eel4 does not seem to playa major rolr bl'('ause whethrr given i.p. or i.g. damage to AL or HL was not signific<:ntly differt'llt. Total hepatiC' blood flow pxprrssed per g of liver tissue is mo­derately deereased in AI. (artrrial blood only) ('omparrd to HL (artrrial + portal blood) (personal observation). 2. ('hanges in drug metabolism. Decreased formation of reactive toxic metabolites could be dm' to :-enraJ factors such as: a decreased activity of cytochromes P 450 dependent enzymps inndn'd in CC\ metabolism, a change in endoplasmic reticulum membrane phospholipid composition, an increased bio-availability of conjugating agents such as glutathionr which normally preYrnts binding of toxic metabolites to macromolecules. 3. changes in sinusoidal cells linkrd to profound disturbances in the delivery of substances to the AL: nutrients, hormonrs. endotoxins (G, 9).

Partial prrvention of hrpatotoxieity was also associated with a gre;~ter liver lobe weight increase suggesting a different eaparity to respond to an aggression. The number of mitoses was not apparently increased. Whatever thr exact mechanisms of hepatoprotection are, this observation desern's further investigation and may help to understand the incidence of hepatic blood fI()w di,tnrbaucps on various conditions including drug toxicity.

Literature

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9. ::\01_\X, .T. P., and D.:-;. (' \'\L\IU, I£ndotoxin, sinusoidal tells anti livt'f injury. In: Progress in Liv('r Dis('as!'s, cds. II. POPPEll and F. ~nL\FFNER; vol, 7. Grune & ~trattol1, New York 1982, pp. 361 ~-;)7Ii.

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