The Effects of Serum from Patients with Acute LiverFailure on the Growth and Metabolism of Hep G2 Cells

Q. Shi, J.D.S. Gaylor, *R. Cousins, †J. Plevris, †P.C. Hayes, and M.H. Grant

Bioengineering Unit and *Department of Chemical and Process Engineering, University of Strathclyde, Wolfson Centre,Rottenrow, Glasgow, and †Liver Unit, Department of Medicine, University of Edinburgh, Royal Infirmary,

Edinburgh, United Kingdom

Abstract: In many bioartificial liver systems currently be-ing designed and evaluated for use in fulminant hepaticfailure, direct contact is required between the patient’sblood and the liver cells in the device. The efficacy of suchdevices will be influenced by the interaction of fulminanthepatic failure (FHF) patient serum with the cells. Wehave found that FHF serum inhibits the growth rate andthe synthesis of DNA, RNA, and protein; disturbs gluta-

thione homeostasis; and induces morphological changes incultured human Hep G2 cells. These interactions shouldinfluence the design of bioartificial liver devices based onproliferating cell lines and indicate the requirement to pre-treat FHF patient plasma to reduce the toxin load. KeyWords: Bioartificial liver—Cytotoxicity of fulminant he-patic failure serum—Hep G2 cells.

The liver is a unique organ that can control itsgrowth according to the demands of the body onhepatic functions (1). The potential for liver regen-eration is considerable and suggests that patientswith fulminant hepatic failure (FHF) might be ex-pected to survive if a temporary detoxification de-vice were available for liver support during the acutephase of the illness. Several bioartificial liver systemsnow on clinical trial support this view (2,3,4). Al-though there is convincing evidence to show the use-fulness of these systems, it is generally agreed thatnone of the bioartificial liver systems currently avail-able can be used as a well defined and practical op-tion for therapy (2,3). This is primarily because theefficacy of the current systems is not great enough toprovide sufficient support for patient recovery with-out liver transplantation.

There are many reasons for this, and both the con-stituents and the configuration of the bioreactorneed to be improved. The former refers to the use ofhighly differentiated hepatocytes and optimal hepa-tocyte mass to provide adequate metabolic supportand the latter to the organization or arrangement of

these cells in a bioreactor to enhance the hepatocytefunctions. However, another factor which could po-tentially diminish the efficacy of any bioartificialliver system is the toxic effect of the patient serumon the hepatocytes in the bioreactor. It has beendemonstrated that serum from FHF patients inter-feres extensively with cellular metabolism. This in-terference includes the inhibition of DNA, RNA,and protein synthesis (5,6) and the loss of cellularintegrity and ion transport (7) as well as actin pre-cipitation (8). Because the detoxification of blood byhepatocytes is essential to all bioartificial liver de-signs and many devices involve direct contact be-tween the patient’s serum or plasma and the cells, itwould be important to investigate the direct interac-tions between FHF serum or plasma and hepato-cytes and their influence on the function of the de-vice.

Hep G2 cells provide a readily available source ofdifferentiated liver cells. The metabolic profile ofHep G2 cells has previously been found to be similarto that of human hepatocytes (9,10), and these cellshave previously been used in an artificial liver device(11,12). The purpose of this study was to investigatethe effects of FHF serum on Hep G2 cells in terms ofcell growth rate, synthesis of macromolecules (DNA,RNA, and protein), alterations in the redox balance

Received March 1998; revised June 1998.Address correspondence and reprint requests to Dr. M. Helen

Grant, University of Strathclyde, Bioengineering Unit, WolfsonCentre, 106 Rottenrow, Glasgow G4 ONW, U.K.

Artificial Organs22(12):1023–1030, Blackwell Science, Inc.© 1998 International Society for Artificial Organs

1023

of the cells (by monitoring the effect on reducedglutathione [GSH] status) and its effects on the ac-tivities of key enzymes involved in the detoxificationof electrophilic metabolites and peroxides.

MATERIALS AND METHODS

MaterialsCell culture reagents, including Dulbecco’s mini-

mum essential medium (DMEM), fetal calf serum(FCS), penicillin-streptomycin, sodium pyruvate,and L-glutamine were purchased from GIBCO, LifeTechnologies, Ltd. (Paisley, Scotland, U.K.). Allother chemicals were products of Sigma ChemicalCo. (St. Louis, MO, U.S.A.) unless otherwise men-tioned. FHF serum was pooled from patients withacute liver failure due to paracetamol overdose. Allpatients were in the intensive therapy unit (ITU) atthe time of sampling and fulfilled the criteria fortransplantation. Samples were taken within 48–96 hof paracetamol overdose, and none of the patientshad any detectable paracetamol in their serums atthe time of sampling. Normal serum was obtainedfrom normal individuals.

Cell cultureHep G2 is a human hepatoma derived cell line,

and it was routinely grown in mono/multilayer cul-tures in DMEM containing 10% (vol/vol) FCS in anatmosphere of 5% CO2 in air. Cells were subcul-tured every 7 days at a split ratio of 1:3 using 0.05%(wt/vol) trypsin and 0.02% (wt/vol) Versene in phos-phate buffered saline (PBS), pH 7.4, to detach thecells.

Cell growth experimentsFor experiments on cell growth in different media,

Hep G2 cells were inoculated in 24 well plates (Co-star, Cambridge, MA, U.S.A.), exposed either to10% (vol/vol) FHF serum, 10% (vol/vol) normal se-rum, or 10% (vol/vol) FCS in DMEM. After thetime periods indicated, the media were removed andstored at −70°C for measurement of lactate dehydro-genase (LDH) leakage. The wells were washed withPBS, and GSH was extracted into 200 ml 10% (wt/vol) trichloroacetic acid (TCA) for 10 min at roomtemperature. Samples were frozen at −70°C. Thewells were washed again with PBS and 150 ml 0.5 MNaOH added to digest protein overnight at 37°C.Samples were stored at −20°C until analysis. Theseeding densities for individual experiments are de-tailed in the results section.

DNA, RNA, and protein synthesisFor the measurement of DNA, RNA, and protein

synthesis, cells were grown in 25 cm2 flasks and ex-posed to media containing either 10% (vol/vol) FHF

serum, 10% (vol/vol) normal serum, or 10% (vol/vol) FCS for 48 h, and then the media were renewedwith 1 mCi of tritiated thymidine, uridine, or leucine(Amersham) for measurement of DNA, RNA, orprotein synthesis, respectively. After another 48 hincubation, the media were discarded, and the flaskswashed with PBS 3 times and then flooded with ei-ther 0.5 M thymidine, uridine, or leucine for 5 min atroom temperature. The cells were washed again withPBS twice and incubated with 10% TCA for 10 minat room temperature. The acid soluble radioactivitywas discarded and 0.5 M NaOH added to solubilizeprotein for 18 h before measuring the incorporationof tritium into the cellular DNA, RNA, and proteinby scintillation counting.

Enzyme activitiesTo measure the effects of serums on enzyme ac-

tivities, Hep G2 cells were allowed to grow to con-fluence (7 days after passage) and then exposed toeither 10% FHF serum, normal serum, or FCS for 48h. Homogenates were then prepared in 2 ml 0.1 Msodium phosphate buffer, pH 7.6, by scraping thecells with a rubber policeman and homogenizingthem using 7–10 strokes of a motor-driven Teflon-glass homogenizer. The homogenates were stored at−70°C until measurement of enzyme activities.

Biochemical determinationsGlutathione-S-transferase (GST) activity was

measured in 50 ml samples of cell homogenate using0.2 mM ethacrynic acid (EA) as the substrate in thepresence of 1 mM GSH as described by Habig andJakoby (13). This substrate is preferentially metabo-lized by the pi isoenzyme of GST, which is known tobe expressed at a high level in tumor cells. Catalaseactivity was measured on 250 ml cell homogenate(14), glutathione reductase on 100 ml (15), andNADPH cytochrome c reductase on 250 ml (16). Theactivities of the selenium dependent and indepen-dent glutathione peroxidases were measured in 20 mlcell homogenate by a modification (17) of themethod of Rister and Baehner (18) using cumenehydroperoxide (total peroxidase activity) or hydro-gen peroxide (selenium-dependent peroxidase activ-ity) as the substrate. Lactate dehydrogenase (LDH)activity was measured in the medium as describedpreviously (19). Protein content was measured bythe method of Lowry (20) after solubilizing themonolayers with 0.5 M NaOH, and the GSH mea-surement was determined by fluorimetric assay (21).

Preparation of samples for scanning electronmicroscopy

Hep G2 cells were grown on glass slides accordingto the same treatment schedule described in the cell

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culture section. Samples were fixed using 2.5% glu-taraldehyde and then dehydrated and coated withgold for scanning electron microscopy (SEM).

StatisticsResults were expressed as mean ± SEM with the

number of experiments in parentheses. Statisticalanalyses were carried out by ANOVA followed byDunnett’s test.

RESULTS

Effects of FHF serum on Hep G2 cell growthThe Hep G2 cell growth rate was significantly in-

hibited in 10% FHF serum during the 4 day cultureas shown in Fig. 1. When inoculated at 105 cells/cm2,a significant difference in growth rate occurred after3 days of culture in medium containing FHF serum.At the endpoint of the experiment, the protein con-tent of Hep G2 cells cultured in medium supple-mented with 10% FHF serum was 3 times less thanthat of those grown in medium containing 10% FCS(73.2 ± 14.6 compared to 219.0 ± 44.2 mg/well, p <0.05, n 4 4). In contrast at the endpoint, the proteincontent of cultures grown in 10% normal serum wassignificantly higher than that of the controls in 10%FCS (311.2 ± 60.5 compared to 219.0 ± 44.2 mg/well,p < 0.05, n 4 4). Cell density at inoculation was animportant factor influencing the magnitude of theeffects of serums. Figure 2 shows the cell growth at104 and 105 cells/cm2 in the different serums. Theeffect of FHF serum was more pronounced at thehigher seeding density.

Effects of FHF serum on GSH content of HepG2 cells

As observed previously (22), the GSH content ofcontrol Hep G2 cells declines with time passage as

the cell growth rate slows down and the culturesapproach confluency. Exposure of Hep G2 cells toFHF serum resulted in a significant increase in theGSH content within 48 h, compared to control cul-tures in 10% FCS or 10% normal serum cultures(Fig. 3). After 4 days the GSH level declined dra-matically to below the levels in control cultures. Therate of GSH content decline was similar at 2 differ-ent densities although, as expected, the GSH levelswere generally higher in the more dense cultures(105 cells/cm2) (Fig. 4). When confluent Hep G2 cellswere challenged with medium containing 10% FHF,the GSH content was significantly increased after 25h culture compared to cells incubated with either10% FCS or normal serum (Fig. 5).

DNA, RNA, and protein synthesis of Hep G2 cellscultured in different serums

The marked inhibitory effect of 4 day exposure to10% FHF serum on the Hep G2 cell DNA, RNA,and protein synthesis is shown in Table 1. Taking thevalues of the FCS group as 100%, the figures from

FIG. 1. The graph shows the total protein content of Hep G2 cellsgrown in media supplemented with 3 different serums. Hep G2cells were seeded at 105 cells/cm2. Cell growth in the 10% FHFserum and normal serum was compared with that in the 10%FCS (*p < 0.05, by ANOVA followed by Dunnett’s test). Resultsare the mean ± SEM of 4 experiments (m: 10% FHF serum, j:10% FCS, d: 10% normal serum).

FIG. 2. The graph shows Hep G2 cell growth in media with 3serums when seeded at 104 cells/cm2 (gray) and 105 cells/cm2

(black) (*p < 0.05, by ANOVA followed by Dunnett’s test, com-pared with the 10% FCS control). Results are the mean ± SEM of4 experiments.

FIG. 3. Shown is the GSH content of Hep G2 cells grown in the3 different serums. The seeding density was 105 cells/cm2 (*p <0.05, by ANOVA followed by Dunnett’s test, compared with the10% FCS control). Results are the mean ± SEM of 4 experiments(m: 10% FHF serum, j: 10% FCS, d: 10% normal serum).

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the FHF group were only 14.5 ± 2.4% for DNA, 46.4± 10.5% for RNA, and 43.1 ± 5.4% for protein syn-thesis. Exposure to 10% normal serum resulted in aslight increase in both DNA and protein synthesiscompared to control cultures.

Enzyme activities in Hep G2 cells cultured indifferent serums

Table 2 illustrates 5 different enzyme activities,including GST, catalase, glutathione reductase, glu-tathione peroxidase, and NADPH cytochrome c re-ductase, in confluent Hep G2 cells cultured in thedifferent media. After 48 h exposure to the differenthuman serums, the activities of these enzymes werenot significantly altered in the Hep G2 cells. Theactivity of LDH in medium containing 10% FHFserum is approximately 20 times higher than that ofmedia containing 10% FCS or 10% normal serum(data not shown). Because of this, it proved difficultto use this method although it would appear thatthere was no significant increase in LDH leakageduring 4 day culture under any of the conditionsused.

Morphological changes in Hep G2 cells cultured inFHF serum

The morphology of Hep G2 cells underwent a dra-matic change once they were put into 10% FHF se-rum. Most of the cells became round and had ahigher tendency to detach from the anchorage sur-face. With increasing time in culture, no cell sheetwas formed to constitute a confluent monolayer, butbatches of aggregates were observed. The mem-branes of the cells became indistinct and the cyto-plasm particulate. Figure 6 shows SEM pictures ofHep G2 cells cultured in different serums for 4 days.

DISCUSSION

When hepatic failure occurs, a wide range of po-tential toxins accumulates in the patient blood. In

FHF, patients develop encephalopathy followed bycerebral edema, which is one of the major causes ofmortality in liver disease (23). There are at least adozen potential toxins involved in the pathogenesisof hepatic encephalopathy, and several hypotheseshave been proposed to explain the pathophysiologi-cal and clinical observations (24). Toxins may origi-nate from either cellular necrosis debris and break-down products released into the blood stream or theaccumulation of an agent or agents that are normallyeliminated by metabolism in the hepatocytes. Para-cetamol overdose is regarded as a classic model forFHF although there may be differences in the bio-chemistry and also in the toxins generated in virallyinduced FHF.

In this study, we have demonstrated the inhibitionof macromolecule synthesis and cell growth in thepresence of FHF serum. Intracellular GSH homeo-stasis was disturbed, but there was no significant ef-fect on the enzyme activities involved in the detoxi-fication of electrophilic species, free radicals, andperoxides.

Previous experimental studies (5,6,25) have dem-onstrated the inhibitory effects of FHF serum ongrowth and macromolecular synthesis in hepato-cytes. Hughes and coworkers investigated the cyto-toxicity of plasma from patients with various types ofliver diseases using a microtoxicity assay system.This group found that plasma from FHF patients wascytotoxic to isolated rabbit hepatocytes (5). Thehighest cytotoxicity values were found in plasmafrom patients with FHF compared to obstructivejaundice and chronic hepatitis (5). Another report byHughes and his colleagues suggested that the inhibi-tory effect of FHF serum on DNA synthesis wasenhanced in hepatocytes from regenerating livers(6). However, they found that protein synthesis by

FIG. 4. The graph shows the GSH content in Hep G2 cells grownin media with 3 different serums when seeded at 104 cells/cm2

(black) and 105 cells/cm2 (gray) (*p < 0.05, by ANOVA followedby Dunnett’s test, compared with 10% FCS control). Results arethe mean ± SEM of 4 experiments.

FIG. 5. Graphed is the GSH content of confluent Hep G2 cellsafter exposure to 3 different media (*p < 0.05, compared with the10% FCS by ANOVA followed by Dunnett’s test). Results are themean ± SEM of 4 experiments (m: 10% FHF serum, j: 10%FCS, d: 10% normal serum).

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14C-leucine incorporation was reduced in hepato-cytes from both normal or regenerating livers in thepresence of serums obtained from normal controland FHF patients. In addition, Haas et al. demon-strated that the DNA content of rat hepatocytes wasdecreased in 20% FHF serum (25). By using rapidlygrowing Hep G2 cells for our experiments, we haveincreased the sensitivity for detecting this type ofinteraction with liver cells compared with the experi-ments previously conducted in nonproliferating pri-mary cultures of hepatocytes.

Although a significant amount of knowledge existson the role of toxins from FHF serum in the devel-opment of encephalopathy (24), little is known abouttheir effects on metabolism. Several groups havetried to define more closely the substances respon-sible for cytotoxic effects. Sewell and coworkers re-ported that serum from patients with FHF inhibitedthe total and ouabain-sensitive sodium efflux in leu-kocytes (7). This led to inactivation of Na-K-ATPaseactivity and could cause the inhibition both ofanabolism and cell division because no molecularsynthesis can occur without energy provision. Thismechanism could also be operating in the liver cells.Hughes et al. pointed out that heating and dialyzingthe patient plasma reduced the cytotoxicity in allcases, and this was most marked in the patient withviral hepatitis and FHF patients (5). Treatment witha charcoal column was also shown to decrease thecytotoxicity caused by FHF serum. However, despiteall these observations, we do not know which com-pounds present in FHF serum exert toxic effects onliver cells. In the present study, it is unlikely that thepresence of paracetamol in the patient’s serum con-tributes to the cytotoxicity because there was no de-tectable paracetamol in the samples. FHF serum isreported to contain high levels of hepatocyte growthfactor (HGF) (26), but this is unlikely to inhibit thegrowth rate of liver derived cells, and we have shownthat it is not cytotoxic to hepatocytes at concentra-tions up to 50 ng/ml (Grant, unpublished data).

Cells, particularly liver cells, are well endowedwith inherent reactions to eliminate harmful stimuli.When cells encounter a limited dose of toxic sub-

stances, i.e., at sublethal levels, the toxic challengeactivates the cellular defense systems and induces anadaptive reaction, which may include gene modula-tion (27). Intracellular GSH plays an important partin this. GSH participates in reactions that detoxifyelectrophilic xenobiotics and endogenous com-pounds and their metabolites, hydroperoxides, or-ganic peroxides, and free radicals, by conjugatingwith them or by acting as a reductant (28). The in-tracellular GSH content often reflects the fate ofpotentially harmful substances, and examination ofGSH status may reveal information on the mecha-nisms of toxicity. Exposure of Hep G2 cells to 10%FHF serum resulted in a significant increase in GSHcontent between 1 and 4 days after subculture. GSHlevels decline in control cultures of Hep G2 cellswith time from subculture, and this is related to thecell growth rate (22). To avoid the effect of cellulargrowth on GSH synthesis, confluent Hep G2 cellswere tested for their ability to respond to FHF se-rum. In this experiment, a significant increase inGSH content was also found after 25 h exposure toFHF serum. The altered GSH homeostasis was notaccompanied by any significant change in the en-zyme activities measured. The alteration in GSHcontent occurred relatively slowly, only becomingevident after 25 h in confluent cultures. There was no

TABLE 1. Effects of serums on the synthesis of DNA, RNA, and protein

Serum DNA RNA Protein

10% normal 122.6 ± 2.4% (5) 105.7 ± 8.8% (8) 139.2 ± 16.5% (8)10% FHF 14.5 ± 2.4% (8)a 46.4 ± 10.5% (4)a 43.1 ± 5.4% (8)a

a p < 0.05, by ANOVA followed by Dunnett’s test. Results are mean ± SEM with the number ofexperiments in parentheses.

Incorporation of 3H-thymidine, -uridine, and -leucine into DNA, RNA, and protein in cultures grownin 10% FCS were taken as 100% values. Significant inhibition of macromolecule synthesis occurred inthe FHF group compared to the FCS control cultures.

TABLE 2. Enzyme activities of Hep G2 cells cultured indifferent serums

10% FCS10% Normal

serum 10% FHF serum

GST 124.15 ± 8.69 113.02 ± 4.12 138.44 ± 11.87Catalase 127.49 ± 3.26 136.02 ± 11.99 129.49 ± 10.74GSSG-R 69.68 ± 29.89 71.43 ± 5.61 49.82 ± 5.69GSH-Ph 3.24 ± 0.69 2.40 ± 0.05 2.83 ± 0.07GSH-Pc 5.82 ± 0.47 5.55 ± 0.58 5.32 ± 0.21Cyto c-R 15.41 ± 4.45 16.33 ± 1.49 13.79 ± 1.13

Enzyme activities are expressed as nmol/min/mg protein andare mean ± SEM of 3 experiments.

GSSG-R: glutathione reductase, GSH-Ph: glutathione peroxi-dase, measured using hydrogen peroxide (selenium dependent),GSH-Pc: total glutathione peroxidase activity using cumene hy-droperoxide as substrate, and Cyto c-R: NADPH cytochrome creductase.

No significant differences were found between the differentserums.

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FIG. 6. Shown are morphologi-cal changes in Hep G2 cells cul-tured in media containing 10%FCS (A and B), 10% normal se-rum (C and D), or 10% FHF se-rum (E, F, and G) for 4 days.Scale bars are on the pictures.

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evidence of depletion or oxidative loss of GSH inany of the experiments so it is unlikely that the FHFserum contains strongly electrophilic reactive toxins,generates free radicals, or causes oxidative stress inthe cells. Previous studies have shown that Hep G2cells responded to abnormal culture conditions andto exposure to toxic concentrations of bile by in-creased GSH content (29,30), and this is thought tobe the result of an activation of cellular defenses. Wehave also shown that exposure of primary cultures ofrat hepatocytes to FHF serum increases their cyto-chrome P450 content and ability to detoxify ammo-nia to urea (31). This has also been interpreted as anactivation of cellular defenses to deal with sublethalconcentrations of toxins in the FHF serum. In addi-tion, Uchino and coworkers have also demonstratedthat the function of primary cultures of pig hepato-cytes does not deteriorate when they are exposed toFHF serum (32).

Several research groups in the field of the bioar-tificial liver emphasize the advantages and impor-tance of direct contact of the patient’s blood orplasma with the hepatocytes in the bioreactor. Mostdevices for artificial liver support are currently basedon nonproliferating primary cultures. Although weand others have shown that FHF serum is inhibitoryto cell growth, this may not be important if devicesare to continue to be based on nonproliferating cul-tures. However, it may become a key issue with therapid advances being made in the generation of dif-ferentiated immortalized hepatocyte cell lines fromanimal and human tissue for application to bioarti-ficial liver devices. Moreover, with increasing con-cern about the presence of viruses in animal tissues,immortalized human hepatocytes are likely to be-come more important in the future. Based on pres-ent evidence, the direct exposure of hepatocytes toFHF patient serum may activate cellular defense sys-tems, causing for example an increase in GSH, cyto-chrome P450, and urea synthesis and thereby in-creasing the efficiency of the bioartificial liverdevice. Therefore, the necessity to pretreat FHF pa-tient plasma to reduce the toxin load is partially de-pendent on the cell type inoculated in the bioreactor.However, the most important issue is to define thenature of toxins responsible for the development ofencephalopathy in FHF patients to optimize the bio-artificial liver system.

Acknowledgments: The authors would like to thank LizGoldie and Gail Connel for help with SEM and photog-raphy. Q. Shi was a visiting scientist funded by JMS Co.,Ltd., Japan.

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