30, 116127

( 19%)

Studies on Fatty Liver with Isolated Hepatocytes II. The Action

of Carbon Tetrachloride on Lipid Peroxidation, Triglyceride Synthesis and Secretion 1



di Patologia Received


Generale, Corso Raflaello 12, 1978,

30, 10126

and in revised form




ALBANO, Torino,


7, 1978

This report describes some effects of poisoning with carbon tetrachloride on hepatocytes in single cell suspension. In isolat.ed liver cells as well as “in v22)o” CC14 stimulates lipid peroxidation, inhibits bot,h protein synthesis and protein and lipoprot.ein secretion and induces fat accumulation within the cells. As the action of CC14 on lipid peroxidation, our data confirm that its increase induced by CC14 depends on the metabolism of this drug by the NADPH-cytochrome P 160 enzyme system. Furthermore, data reported here suggest that the onset of the CClr-induced decrease of lipoprotein secretion is due to a derangement of the secretory pat,hway.

INTRODUCTION It is widely known that carbon tetrachloride is an important model agent for studying the pathogenesis of liver injury. The biochemical mechanisms by which Ccl, exerts its toxic effects have been studied extensively, “itz uivo” and “in vitro” (see Recknagel, 1967, and Slater, 1973, for reviews). A single dose of the toxic agent produces centrilobular necrosis and fatty degeneration in the liver. The latter event has been related to an impairment of protein synthesis with subsequent failure of lipoprotein formation and secretion (Robinson and Seakins, 1962). However, several drugs produce a strong and rapid inhibition of protein synthesis and a slower accumulation of fat in the liver (Dianzani and Gravela, 1975) ; this may depend on tho presence jvithin the liver cells of preformed lipid-acceptor protein. In the case of CCL, fatty accumulation can be detected within the first hour of poisoning (Schotz and Recknagel, 1960). This suggests that in CC14-induced fatty liver the earliest derangement involves mechanisms other than inhibition of protein synthesis, e.g. coupling of triglyceride, phospholipid and lipid-accept’or protein to produce lipoprotein, or the intracellular transport of lipoprotein into secretory vesicles and its discharge outside the liver cells through the microtubular system. The CCL-induced alteration of these different steps of lipoprotein secretion could be related to the peroxidative decomposition of structural membrane lipids. In fact, it is well known that an early effect of CCL dosing is an enhanced 1 This work was supported by a grant from the Consiglio Nazionale delle Ricerche, Roma, Italy. 116 0014-4800/79/010116-12$02.00/O Copyright All rights

0 1979 by Academic Press, Inc. of reproduction in any form reserved.





pcroxidation of mcmbrano lipids, as indicated by the dctcction of conjugated diencs in microsomal lipids (Rao and Rccknagrl, 1968). The lipoperoxidation leads to the formation of many products, mainly aldchydcs and ketoaldehydes. Malondialdehydc determination has usually been taken as a measure of lipid peroxidation. Moreover, CC14 could impair the lipoprotein accretion by direct binding of its metabolitcs to the liver lipids and proteins. This binding can be detected shortly after CCL administ,ration (Reynolds, 1963) and its importance as a mechanism in CCL-induced cell injury has been stressed by several authors (McLean, 1967; Cignoli and Castro, 1971; Gillette et al., 1974). In our opinion, the USCof isolat’cd liver cells could elucidate some aspects of the pathogenesis of the CCL-induced liver damage. In previous work, using isolated rat hepatocytes exposed to different drugs, we could draw a distinction between the inhibition of lipoprotein secretion dependent on an impairment of protein synthesis and that induced by a derangement of the secretory system of the hepatocyte (Gravela et al., 1977). We, therefore, studied in isolated liver cells the cffcct of CCL on lipid pcroxidation as well as on protein and triglyceride synthesis and secretion. Our result’s suggest that the onset of the CCL-induced decrease of lipoprotein secretion is due to a derangement of the secretory pathway. MATERIALS



Male rats of Wistar strain, 200 to 250 g in weight, were used. They were maintained on a semi-synthetic diet, devoid of any antioxidant (Piccioni, Brescia, Italy) and water ad Zibitum. Operations were performed between 10 AM and 1 PM. All chemicals were of reagent grade and were obtained from the following sources. Collagenasc (CLS, type IV), Wort’hington Biochemical Corp., Freehold, N.J. ; Cycloheximide, HEPES, colchicine and amino acids, Sigma Chemical Co. St. Louis, MO. ; 2-dicthyl-aminocthyl-2,2-diphenylvalerate (SKF-525A), Smith, Kline, and French Laboratories Ltd., Herts, U.K.; radiochemicals, The Radiochemical Centre, Amersham; other chemicals, BDH Chemicals Ltd., Poole, U.K. Rcagcnt kits from Biochcmica Test Combination, Boehringcr, Mannheim, Germany, were used for the mcasurcmcnt of lactate dchydrogenasc, glutamat,coxalacetate transaminase and glutamate-pyruvatc transaminasc. The composition of the buffers and the medium used, respectively, in the procedure for liver cell isolation and incubation were the following. Saline buffer A contained: 0.143 d1 NaCl; 7 rnA1 KCl; 10 mM HEPES-NaOH buffer pH 7.4. Saline buffer B contained : 0.1 .!lf NaCl ; 7 mM KC1 ; 5 mM CaClz ; 50 mA4 HEPES-NaOH buffer pH 7.6. Medium C contained: 60 mM NaCl; 40 mN KC1 ; 50 mM HEPES-NaOH buffer pH 7.4 ; 1 mM CaC12; 2 mM MgS04 ; 1 mdlr Na2HP04 ; 5 mM glucose; 0.58 rnnil amino acid mixture. The latter contained : 0.05 rnM each arginine, aspartic acid, glycine, glutamic acid, glutamine, leucine and lysine ; 0.02 mM each alanine, asparagine, phenylalanine, isoleucine, serine, histidine, threonine, valine and taurine; 0.01 mM each cysteine, methionine, proline, tyrosine, and tryptophan. Preparation of Isolated Hepatocytes Hepatocytes were isolated by the collagenase perfusion method described in a preceding paper (Gravela et al., 1977). At the suggestion of other authors



(Schreiber and Schreiber, 1975; Scglcn, 1976) the following modifications were incorporated into the method: the livers were first perfused for about 10 min with warm (37°C) saline buffer A; the perfusion rate was 20 to 25 ml/min. Then the livers were perfused for about 10 min with warm (37°C) saline buffer B containing 0.050% collagenase, at a perfusion rate of 10 to 15 ml/min. Livers were then removed, gently dispersed in 50 ml of medium C, and incubated for 10 min at 37°C in a rotary evaporator. The cell suspension was first diluted with 200 ml of medium C, then filtered through a 200 pm mesh and centrifuged at 2009 for 4 min. The pellet was suspended in medium C and the cells counted with a hemocytometer, then diluted with medium C to the cell concentration hereafter described. Cell yields ranged from 200 to 300 million cells for liver, and 90 to 970j0 of hepatocytes were viable as measured by trypan blue exclusion test (Jeejeebhoy et al., 1975). The hepatocyte suspensions were completely free of blood and Kupffer cells. When the liver cells were grown to a monolayer the results indicated an absence of fibroblasts in the above mentioned cell suspensions (Poli, G., unpublished). CC&-poisoning

of Isolated Hepatocytes

The hepatocyte suspension was diluted with medium C to 8 X lo6 cells/ml. Aliquots of 3 ml of the suspension were placed into the main compartment of 50 ml flasks, fitted with a center well and closed with a screw cap. CCL was added in the center well and allowed to diffuse in the closed system. Flasks were incubated for 60 min at 37°C in a Dubnoff water bath. Control cells were incubated in the same way, but without CCl4. By adding amounts of 14C-Ccl4 ranging between 2.5 and 20 ~1 the actual concentration of the toxic agent in the cell suspension was determined. The dissolved radioactivity was about 1: 200 of the total added radioactivity. Excepting in preliminary experiments, which will be reported in the Results section of this paper, the amount of Ccl, added in the center well of the flasks was 7.5 ~1. With 7.5 ~1 i4C-CC14 the actual concentration of CC14 in the cell suspension was about 20 rig/ml. The lipid-bound radioactivity, determined according to Rao and Recknagcl (1969), corresponded to about 5 ng CCL per lipid from 1 g hepatocytes. This value did not change significantly in samples taken after different times of incubation ranging between 5 and 60 min. It is noteworthy that the lipid-bound radioactivity, taken as a measure of the covalent binding of CC14 metabolites, we found is quite similar to that observed after poisoning “in viva” (Rao and Recknagel, 1969). Deternair~ation

of Malondialdehyde

Malondialdehydc production was estimated by measuring the thiobarbituric acid-reacting compounds (Bcrnheim et al., 1948). After the above incubation, suspension aliquots corresponding to 2 X lo6 cells were added to 10% trichloroacetic acid (TCA) and water to give a final volume of 3 ml and final 5oj, TCA concentration. After centrifugation, 1.5 ml aliquots of the supernatants were treated with the same volume of 0.67y0 thiobarbituric acid, incubated in boiling water for 10 min and made alkaline with KOH (final concentration 0.29 M). Absorbance at 543 nm was determined using a Beckman ACTA III spectrophotometer.





The eventual damage of liver cells after incubat8ion with diffcrcnt amounts of CCL was evaluated by measuring the release of lactate dchydrogenase (LDH), glutamate-oxalacetate t,ransaminase (GOT) and glutamate-pyruvatc transaminase (GPT) with kit proccdurcs (Biochcmica Test Combination). Ccl,poisoned and control cell suspensions (1 ml each, 8 X lo6 cells) were centrifuged at 600g for 4 min. Supernatant aliquots of 15 to 30 ~1 were used for enzyme determination. The opt,ical density at 340 nm \vas detcrmincd using a Beckman ACTA III spectrophotomet’er, and the extracellular enzyme activity was expressed as percent of total (intracellular plus extracellular) enzyme activit,y, determined after cell dest’ruction with 0.5y0 Triton X 100.

l%oteit~ ad Lipid Metabolism Studies. These were carried out after the ti0 min preincubation with or without CC14 above described. The cells, suspended in medium C, were diluted 1: 10 with medium C and centrifuged at 2OOgfor 4 min. The pellets were resuspended in Ham’s F-12 medium (Ham, 1965), containing 10% horse serum and 0.17c penicillin G, to give a concentration of 4 X 10” cclls,/ml. Aliquots of 1 ml were placed into culture dishes (33 X 10 mm) in t,he presence of 14C-valine or of a 14C-fatty acid mixture. In all t#hc cxptriments hereafter described, the zero-time control values were determined and subtracted from the observed values. Protein synthesis and secretion. CCll-poisoned and control cells were incubated at 37°C with 14C-valine (U-W)-L-valine, specific activity 270 mCi/mmol) 0.25 &Ji/sample. After different times of incubation the reaction was stopped by diluting the cell suspension with 4 ml of 0.9y0 SaCl containing unlabeled 10 mM L-valinc. The samples were immcdiaMy ctntrifuged (6OOg for 4 min). Proteins in pellets or in supcrnatants were precipitated with 57, TCA, then rinsed twice again with 5% TCA. The protein digestion and radioactivity mcasurement were carried out as previously described (Gravela et al., 1977). Triylyceride syttthesis a,rd secretion. CC&-poisoned and control cells were incubat,ed at 37°C wit,h 0.2 ml of a 14C: W-fatty acid mixture The latter contained: 0.15 21 NaCl; 47o albumin; 1 ml11 (0.33 pCi/ml) each sodium oleatc, palmitate and stearate ; 0.1 mM (0.066 &i/ml) sodium arachidonate ; 1 mM phosphorylcholine. Sodium hydroxide was used to adjust the pH to 7.4. Fatty acids were complexrd with albumin according to Capuzzi et al. (1974). After different times of incubation at 37°C the cc11suspension was diluted with 4 ml of 0.9% NaCl and immediately centrifuged at 6OOg for 4 min. Intracellular triglycerides and extracellular lipoprotein triglycerides were separated, purified and processed for radioactivit,y dctcrmination as clsc\\herc described (Gravela et al., 1977). Protein. awl triglyceride secretion ft~m prelabeled hepatocytes. For these studies cells were suspended, after the isolation procedure, in Ham’s F-12/horse serum medium, in the concentration of lo7 cells/ml. Cell protein was labeled by incubating IO ml of hcpatocytcs with 20 PCi of 14C-valine for 60 min at 37“C. Then radioactive incorporation was stopped by diluting the ccl1 suspension with 100 ml of medium C containing, in addition, 10 mM unlabeled L-valine and 10 pg/ml of cycloheximide. The diluted suspension was centrifuged at 4009 for 4 min. Cell triglyceride was labeled by incubating 10 ml of hepatocytes with 5 ml of the




14C: ‘%-fatty acids mixture described above. After 60 min incubation at 37°C the cell suspension was diluted with 100 ml of medium C containing 10 pg/ml of cyclohcximide and then centrifuged as above. Either ‘“C-valinc labeled or t’hc 14C-fatty acids labeled hcpatocvtcs wcrc suspended in medium C containing 10 rg/ml of cycloheximidc. It has been shown (Schreiber and Schreiber, 1975) that cycloheximide almost completely stops prot,ein synthesis in isolated hepatocytes without influencing the rate of secretion of prelabeled protein. Cell suspensions were then diluted with the same medium in order to contain 5 X 10” cells/ml. Aliquots of 2 ml of the suspensionswere poured into the flasks above described, with Ccl, (7.5 ~1, in the center well of the flasks), or colchicine (50 PM final concentration), or without any addition. After 15 min of prrincubation at 37°C the content of somr flasks was immediately centrifuged, to determine the zero time secretion in the supernatants; the other flasks were further incubated at 37°C for 10, 20, 30, and 40 min. At the end of the incubation, cell suspensions were immediately centrifuged. Protein and lipoprotein t’riglyceride in supernatants were purified and processed for radioactivity measurement as described elsewhere (Gravela et al., 1977). RESULTS General Features of the System Preliminary experiments have been carried out in order t’o decide the best conditions for poisoning of isolated liver cells with Ccl,. Weddle et al. (1976), working with isolated rat liver cells, have shown that Ccl, directly suspended into the cell medium (1 to 10 PI/ml) did stimulate lipid peroxidation. These authors, however, did not study the metabolic activity of the poisoned cells. We have found that, when CC14 is added to the hepatocyte suspension according to the mentioned method, in amount of 1 to 2 pi/ml, there is not only a dramatic decrease in trypan blue exclusion as reported by Weddle et al. (1976), but also an increase in the extracellular release of LDH, GOT, and GPT. We decided, therefore, to incubate hepatocytes in closed flasks, where CCL was added in a center well and allowed to diffuse in the system. In this way, very low concentrations of CCL were recovered from the incubation mixture (see the Materials and Methods section of this paper). In a previous work (Gravela et al., 1977) we studied the protein and lipid metabolism of the isolated hepatocytes suspended in a complete medium (Ham’s F-12/horse strum), in agreement with data reported by Jeejeebhoy et al. (197.5). Some experiments have suggested the suspension of the liver ccl1 in medium C instead of Ham’s F-12/horse serum medium during the CCli-poisoning step, and to use the second medium only for the protein and lipid metabolic studies. A typical experiment, reported in Table I, showed that there is no evidence for CCL stimulation of malondialdehyde production in the cells when they are suspended in Ham’s F-12/horse serum medium. On the cont,rary, this stimulation occurred in hepatocytes suspended in medium C. The dependency of CC&induced lipid peroxidation on the metabolism of this drug by the NADPH-cytochrome P 450enzyme system of the cell was confirmed by the inclusion in the medium of 50 PM SKF-525A, which caused a significant decrease in the malondialdehyde production due to Ccl, (see Table I>. It is, in





JIalondinldehyde Cell

Noue Ccl, SKF-52.5A HKF-525A a Absorbance hepatocytes. * 7.5 PI/flask


in Isolated

Hepatocytcs nicdium

0.09ci 0.240 0.092 0.126

+ CClr

at 543 nm




of the



f zk * +




Hcpatocytes in Ham’s F12serum medium


0.015 0.035 0.003 0.011

0.101 0.109



zk 0.006 + 0.014




50 p*M SKF-525A.

fact, known that SKF-525A causes inhibition of drug metabolism by its intcraction in the neighborhood of cytochrome PJ50 (Slater and Sawyer, 1971). Data reported in Fig. 1 show that t’he highest stimulation of malondialdehyde formation is achieved with 73 ~1 CCI, per flask. This amount of CC14 does not induce evident change in cell viability as checked bot.h by trypan blue exclusion and enzyme release (see Fig. 2). Amounts of CC14 higher than 10 ~1 do not stimulate further lipid peroxidation, but do induce extensive cell damage. These findings were confirmed by studies on triglyceride synthesis and secretion reported in Table II, where it is evident that cell-poisoning wit’h 15 ~1 of CC14 induces a strong inhibition of fatty acid incorportation into ~11 lipids. In hepatocytes poisoned with 2.5 or 7.5 ~1 of CCL, however, fatty acid incorporation was similar to that observed in untreated cells. Moreover, in hppatocytcs poisoned with 7.5 ~1 of CCL there was a significant decrease in triglyceride secretion, mainly evident after 2 hr of incubation. For these reasons in all studies hereafter reported we poisoned hepat’ocytcs with 7.5 ~1 of Ccl?.

/ +








PI CCl,/flask FIG. 1. Effect of increasing amount, of CClr on malondialdehyde production in isolated hepatovytes suspended in medium C. Aft,er incubation of the hepatocytes for 60 min at 37°C in presence or in absence of CClr, cell suspension aliquots (2 X 10” cells) were used. Values on the ordinate are absorbance at 543 nm of the thiobarbituric acid-react,ing compounds produced/h by 10” hepatocyies [mean of tJx= experiments *SD).







10 pl C Cl,



/ flask

FIG. 2. Effect of increasing amount of CC14 on hepatocyte damage estimated by trypan blue stainability and by enzyme release. After incubation of t’he hepatocytes for 60 min at 37°C in presence or in absence of CCla, cell suspension aliquots were used. Values on the ordinate are percent of trypan blue stained cells, or percent of extracellular enzyme activity over total intracellular and extracellular enzyme activity determined after cell destruction induced by 0.57, Triton X 100.

E$ect of CCL-Poisoning on Protein and T$$yceGle

Synthesis and Secretion

It has been shown that in the liver of CCL-poisoned rats an early inhibition of protein synthesis occurs (Smuckler and Benditt, 1965; Gravela and Dianzani, 1970). Hepatocytes also show a marked impairment of protein synthesis after CCL-poisoning in our ‘(ill, z&o” system (see Fig. 3). Extracellular protein-bound radioactivity remains very low in the CC14 group. This finding confirms the absence of nonspecific release of intracellular protein due to CCL. TABLE Triglyceride Cell pretreatmerit”

60 min

Synthesis incubation radioactivityb

Intracellular None CCL 2.5 /J CCI, 7.5 p1 CCL 15 pl

18,426 17,983 20,124 6,381

f 2,132 zk 1,115 f 568 f 58

and Secretion

in Isolated


Extracellular 2,235 2,192 1,040 403


f 379 f 413 f 195” f 181


120 min

Total 20,661 20,175d 21,164d 6,784

incubation radioactivity*

Intracellular 32,361 32,486 37,319 9,846

f 2,627 zk 1,780 f 920c f 166


Extracellular 4,756 4,935 2,268 577

f 662 Y!Z 323 f 426 3~ 320

Total 37,117 37,421d 39,587d 10,423

0 Hepatocytes, suspended in medium C, have been preincubated for 60 min at 37” in the presence, or in the absence, of CCL. Then they have been rinsed, suspended in Ham’s-FlZ/horse serum medium, and incubated for 60 or 120 min at 37°C in the presence of W-fatty acids. * Numbers are cpm/4 X 10” cells (mean of two triplicate experiments &SD). c By Student’s t test, the difference between the controls and the treated is significant at P

Studies on fatty liver with isolated hepatocytes. II. The action of carbon tetrachloride on lipid peroxidation, protein, and triglyceride synthesis and secretion.

EXPERIMENTAL AND hIOLECULAR PATHOLOGY 30, 116127 ( 19%) Studies on Fatty Liver with Isolated Hepatocytes II. The Action of Carbon Tetrachloride...
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