effect of tauroursodeoxycholic acid on bile acid-induced apoptosis in primary human hepatocytes

European Journal of Clinical Investigation (2000) 30, 203±209 Paper 615

Effect of tauroursodeoxycholic acid on bile acid-inducedapoptosis in primary human hepatocytes

C. Benz, S. AngermuÈ ller*, G. Otto², P. Sauer, W. Stremmel and A. Stiehl

Department of Medicine and *Department of Anatomy and Cell Biology, University of Heidelberg, and ²Department of

Surgery, University of Mainz, Germany

Abstract Background/aims The accumulation of endogenous bile acids contributes to hepato-

cellular damage during cholestatic liver disease. To evaluate the potential role of apoptotic

cell death due to increased concentrations of bile acids, primary human hepatocytes were

treated with hydrophobic and hydrophilic bile acids. Because the Fas receptor±ligand system

may mediate apoptosis in human liver cells, the effect of toxic bile acids on hepatocellular Fas

receptor expression was evaluated.

Materials and methods Primary human hepatocytes were incubated with 50 and 100 mM

glycochenodeoxycholic acid (GCDCA) and co-incubated with equimolar concentrations

of tauroursodeoxycholic acid (TUDCA). To evaluate cytolytic and apoptotic effects,

morphological alterations, hepatocellular enzyme release, nuclear DNA fragmentation

and hepatocellular Fas receptor expression were evaluated.

Results Apoptotic cell death was signi®cantly increased after exposure to 50 mM GCDCA.

Bile acid-induced apoptosis was not accompanied by hepatocellular Fas receptor over-

expression. Tauroursodeoxycholic acid reduced apoptosis, as indicated by a signi®cant

reduction of oligonucleosomal DNA cleavage. Fas receptor expression was not signi®cantly

affected by tauroursodeoxycholic acid. At higher concentrations, direct cytolytic cell

destruction was observed.

Conclusion Primary human hepatocytes represent a suitable model to study bile acid-

induced apoptotic cell death. In these hepatocytes, already low bile acid concentrations

might induce apoptotic cell death, which is not triggered by hepatocellular Fas receptor

overexpression. Apoptotic DNA fragmentation was signi®cantly reduced by co-incubation

with tauroursodeoxycholic acid. The reduction of bile acid-induced apoptosis by ursodeoxy-

cholic acid and its conjugates may contribute to the bene®cial effects of these hydrophilic bile

acids used for medical treatment of several cholestatic liver diseases.

Keywords Apoptosis, bile acids, human hepatocytes, tauroursodeoxycholic acid.

Eur J Clin Invest 2000; 30 (3): 203±209

Introduction

In patients with cholestatic liver disease, hepatocellular

damage may be aggravated by increased bile acid concen-

trations [1]. At high bile acid concentrations hepatocellular

damage may be due to the detergent effects of bile acids

leading to direct destruction of cell membranes [2]. In most

patients, however, such high bile acid concentrations are

not observed [3] and therefore hepatocellular damage at

low bile acid concentrations is of special interest. In pri-

mary rat hepatocytes, short exposure to low concentrations

of toxic bile acids was found to induce apoptotic cell death

[4±7], which was reduced by co-administration of ursode-

oxycholic acid (UDCA) and its conjugates [4,7]. More-

over, in mice it has been suggested that the Fas system may

be involved in bile acid-induced hepatocellular apoptosis

[8]. As considerable differences in bile acid metabolism

between rodents and humans exist [1], studies in humans

seem essential. In primary human hepatocytes, the bene-

®cial effect of UDCA on bile acid-induced cytolysis has

Q 2000 Blackwell Science Ltd

Department of Medicine (C. Benz, P. Sauer, W. Stremmel, A.

Stiehl) and Department of Anatomy and Cell Biology

(S. AngermuÈ ller), University of Heidelberg, Heidelberg,

Germany; and Department of Surgery , University of Mainz,

Mainz, Germany (G. Otto).

Correspondence to: Postal address: Christine Benz, MD,

Medizinische UniversitaÈtsklinik, 69115 Heidelberg, Germany.

Fax: 06221±565687

Received 10 June 1999; accepted 6 November 1999

204 C. Benz et al.

been shown [9], but its possible effect on apoptosis has not

been studied.

In hepatobiliary tissue, two ligand-dependent pathways

triggering apoptosis (Fas/apo-1 and TGF-b) have been

identi®ed [5]. In mice, the activation of the Fas receptor±

ligand system, which has a proven sequence identity with

the apo-1 system [10], has been found to induce fulminant

liver failure [11]. In humans, a possible role of Fas-

mediated apoptosis has been suggested in the patho-

physiology of viral hepatitis, fulminant liver failure, alcoholic

liver damage and primary biliary cirrhosis [12±15]. In the

present study, the possible effects of bile acids on hepato-

cellular Fas receptor expression were evaluated. Corre-

sponding to our previously described experiments in

primary rat hepatocytes [7], we exposed primary human

hepatocytes to low concentrations of the hydrophobic bile

acid glycochenodeoxycholic acid (GCDCA). Taurourso-

deoxycholic acid (TUDCA) was used to study the poten-

tially bene®cial effects of hydrophilic bile acids, because it

represents the most hydrophilic conjugate of UDCA [16],

which has been suggested to be more protective than the

glycine conjugate [17]. Moreover in several European

countries TUDCA is on the market for the treatment

of cholestatic liver diseases. Based on our previous results

in primary rat hepatocytes [7], co-incubation experi-

ments were performed at equimolar concentrations of

hydrophobic and hydrophilic bile acids.

Materials and methods

Cell cultures

Primary human hepatocytes were prepared from healthy

liver tissue obtained from patients undergoing partial

hepatectomy for metastatic liver disease. Informed consent

was obtained in all cases. Histological examination con-

®rmed that tumour-free tissue was used. Patients with liver

diseases other than cancer (e.g. cirrhosis) were excluded.

Hepatocytes were isolated by using the two-step collagenase

perfusion method of Seglen [18], with a viability exceeding

80% according to the trypan blue exclusion method. The

culture medium used was Williams' medium E containing

5% foetal calf serum (Gibco, Nunc, Wiesbaden, Germany).

Cells were seeded at a density of 1 ´ 105 cells cmÿ2 on plastic

tissue-culture dishes. Two hours after plating the medium

was replaced and bile acids were added. For seeding and

maintenance of cells, Williams' medium E was supplemen-

ted with 0´066 mM insulin (Serva Biochemicals, Heidelberg,

Germany), 0´1 mM glucagon (Eli Lilly, Giessen, Germany),

0´1 mM triiodothyronine (Serva Biochemicals), 5 mM gluta-

mine (Flow Laboratories Gmbh, Meckenheim, Germany),

37 mM inosine (Serva Biochemicals), 10 mg mLÿ1 gentami-

cin (Flow Laboratories GmbH), 100 mg mLÿ1 streptomy-

cin (Flow Laboratories GmbH), 100 mg mLÿ1 penicillin

(Flow Laboratories GmbH) and 20 mM HEPES (Flow

Laboratories GmbH). Cultures were incubated at

37 8C in 5% CO2 and 95% air. To exclude signi®cant

spontaneous apoptosis, all experiments were stopped

after 4 h of incubation.

Bile salts

GCDCA was purchased from Sigma Chemical Co. (St

Louis, MO). TUDCA was purchased from Calbiochem

Novabiochem Corporation (La Jolla, CA).

Enzyme release

Isolated human hepatocytes were plated (1 ´ 106 cells

plateÿ1) and incubated with 50 and 100 mM concentrations

of GCDCA, TUDCA and the combination of GCDCA

and TUDCA at equimolar concentrations. Incubation with

Williams' medium E was used for control experiments.

After treatment for 4 h, the tissue culture supernatant

(1 mL) was harvested and assayed for enzymatic activity

aspartate-aminotransferase (AST) using a commercially

available standard kit (Boehringer Mannheim, Mannheim,

Germany).

In situ dUTP nick-end labelling technique (TUNEL

technique)


plateÿ1) on uncoated plastic culture dishes. Cells were

treated with 0 (for control cells), 50 and 100 mM concen-

trations of GCDCA. Co-incubation experiments were

performed by exposing the cells to equimolar concentra-

tions of GCDCA and TUDCA. After 4 h of treatment, cells

were washed carefully in phosphate-buffered saline (PBS)

and ®xed with methanol±acetone (1 : 1) solution for 2 min.

Nuclear DNA fragmentation was demonstrated by enzy-

matic nick-end labelling using the TUNEL reaction (In

Situ Cell Death Detection Kit, AP; Boehringer Man-

nheim), which was performed according to the manufac-

turer's instructions. Single DNA strand breaks were

identi®ed by labelling free 3-OH termini with modi®ed

nucleotides (dUTP nick-end labelling) and cells were

counterstained by haematoxylin.

Electron microscopy

Primary human hepatocytes were incubated as described

above and processed as follows. After treatment, cells were

carefully washed in 150 mM Pipes (Piperazine-N,N-bis

2-ethanesulphonic acid) and ®xed for 30 min with 1´5%

glutaraldehyde in 100 mM Pipes, pH 7´4. Hepatocytes

were post-®xed with reduced osmium containing 1% aqu-

eous osmium tetroxide and 1´5% potassium ferrocyanide,

dehydrated in graded ethanol and transferred to hydroxy-

propyl methacrylate. Tissue culture dishes were then

poured out with Epon 812. Ultrathin sections were

counterstained with lead citrate for 1 min and with uranyl

Q 2000 Blackwell Science Ltd, European Journal of Clinical Investigation, 30, 203±209

Bile acids and cell damage in human hepatocytes 205


acetate for 3 min, and examined in a Philips EM 301

electron microscope.

DNA fragmentation assay

Quanti®cation of bile acid-induced DNA cleavage was

achieved by measuring oligonucleosome-bound DNA frag-

ments, using a cell death detection enzyme-linked immu-

nosorbent assay (ELISA) kit (Boehringer Mannheim).


plateÿ1) and incubated with GCDCA (50 and 100 mM),

and with unsupplemented Williams' medium E for control

experiments. To evaluate the possible hepatoprotective

effect of TUDCA, co-incubation experiments with equi-

molar concentrations of GCDCA and TUDCA were per-

formed. After 4 h of incubation, cells were harvested and

the cytosolic fraction (13 000 g supernatant) of cells was

used as an antigen in a sandwich ELISA, with a primary

anti-histone antibody coated to the microtitre plate and a

secondary anti-DNA antibody coupled to peroxidase.

From the absorbance values, the percentage of fragmenta-

tion in comparison with controls (untreated hepatocytes)

was calculated according to the following formula:

apoptosis index �

absorbance of sample cells ÿ absorbance of blank

absorbance of control cells ÿ absorbance of blank

Fas/apo-1 quantitative assay

Quanti®cation of Fas receptor expression was performed

with the use of a commercially available ELISA (Fas/apo-1

Quantitative Assay; Dianova, Hamburg, Germany), pro-

viding a primary monoclonal anti-Fas antibody coated to

the microtitre plate and a secondary detector antibody. The

detector antibody is biotinylated, which allows its binding

by horseradish peroxidase-conjugated streptavidin. Quan-

ti®cation was achieved by the construction of a standard

curve using known concentrations of Fas antigen (provided

lyophilized).

After plating (1 ´ 106 cells plateÿ1) and seeding, cells

were incubated with GCDCA (50 and 100 mM) and with

unsupplemented Williams' medium E for control experi-

ments. To evaluate the possible hepatoprotective effect

of TUDCA, co-incubation experiments with equimolar

concentrations of GCDCA and TUDCA were performed.

After 4 h of treatment, cells were harvested and the cytosolic

fraction (13 000 g supernatant) was used to perform the Fas

antigen assay according to the manufacturer's prescription.

Statistical analysis

Data were expressed as mean 6 SD. Statistical signi®cance

was evaluate using the Mann±Whitney test. P-levels of less

than 0´05 were considered to be statistically signi®cant.

Results

Enzyme release

Exposure to 100 mM GCDCA was followed by a signi®cant

increase of AST release (P<0´05; n� 8). In contrast, treat-

ment with 50 mM GCDCA for 4 h did not signi®cantly alter

AST release compared with control cells (n� 8) (Table 1).

Co-incubation with equimolar concentrations of TUDCA

for 4 h did not signi®cantly reduce hepatocellular enzyme

release induced by GCDCA (Table 1).

In situ nick-end labelling technique

Morphological evaluation after treatment of primary

human hepatocytes with 50 mM GCDCA revealed pre-

served cell membranes. However, in contrast to control

cells, many cells showed extensive cytoplasmic vacuoles

(Fig. 1a). Nuclear DNA strand breaks indicating apoptotic

cell death were visualized using the TUNEL reaction.

Scattered cells were characterized by TUNEL-positive

marginated nuclear chromatin and by compaction of the

cytoplasm (Fig. 1b). Hepatocellular damage following

co-incubation with 50 mM GCDCA and 50 mM TUDCA

was reduced compared with treatment with GCDCA

alone. However, many cells showed cytoplasmic vacuoles

and the beginning of cytoplasmic condensation (Fig. 1c).

After treatment with 100 mM GCDCA, hepatocellular

damage was characterized by entirely TUNEL-positive

nuclei and by extensive membrane defects, indicating

direct cytolytic cell destruction (Fig. 1d).

Electron microscopy

Ultrastructural evaluation after treatment with 50 mM

GCDCA revealed morphological alterations of variable

degrees. As depicted in Fig. 2(a), many cells showed

cytoplasmic lipid vacuoles. Corresponding to the ®ndings

of light microscopy, the predominant morphological altera-

tions due to apoptosis were characterized by cellular

shrinkage and by the condensation and margination of

nuclear chromatin. These alterations were accompanied

Table 1 Hepatocellular enzyme release after incubation of

1 ´ 106 cells plateÿ1 for 4 h with 50 and 100 mM concentrations

of bile acids (n�8)

AST

Mean (IU Lÿ1) SD

Control 17´4 3´9

GCDCA 50 mM 19´5 3´9

GCDCA 50 mM� TUDCA 50 mM 19´8 5´9

GCDCA 100 mM 26´6 * 6´3

GCDCA 100 mM� TUDCA 100 mM 25´3 5´9

*Signi®cantly different from the control value with P<0´05.

206 C. Benz et al.

by the formation of blebs of the nuclear membrane and by

the aggregation of cytoplasmic organelles, particularly the

mitochondria and the rough endoplasmic reticulum (Fig.

2b). After co-incubation with 50 mM GCDCA and 50 mM

TUDCA, cytoplasmic and nuclear alterations were less

prominent (Fig. 2c). Following short exposure to 100 mM

GCDCA, primary human hepatocytes showed apoptotic

nuclei but also important signs of cytolytic cell destruction

(Fig. 2d).

DNA fragmentation assay (cell death ELISA)

Incubation with 50 mM GCDCA was followed by signi®-

cantly increased oligonucleosomal DNA cleavage com-

pared with control cells. The DNA fragmentation index

increased from 1 to 1´6 6 0´4 (mean 6 SD; n� 8; P <0´05)

(Fig. 3). Higher GCDCA concentrations (100 mM) caused

no further increase of oligonucleosomal DNA cleavage

(Fig. 3). Co-incubation with equimolar concentrations of

TUDCA signi®cantly reduced GCDCA-induced DNA

fragmentation, approaching the values of the control cells

(P <0´05; n� 8) (Fig. 3).

Fas/apo-1 quantitative assay

Incubation with GCDCA (50 and 100 mM) did not

enhance Fas antigen expression compared with control

cells (n�8; P >0´05) (Fig. 4), and co-incubation with

equimolar concentrations of TUDCA did not signi®cantly

reduce Fas antigen expression (n�8; P >0´05; Fig. 4).

Discussion

Our previously published studies evaluated the time- and

concentration-dependency of bile acid-induced apoptosis

and cytolysis in primary rat hepatocytes [7]. Regarding the

differences in bile acid metabolism between rat and human

[1], the possible role of apoptosis and cytolysis in chole-

static liver disease had to be investigated using primary

human hepatocytes. At very high bile acid concentrations

of up to 500 mM GCDCA, which are found in the serum of

patients with prolonged severe mechanical cholestasis [1],

the subsequent hepatocellular injury has been attributed to

the direct membrane-damaging action of bile acids [1]. In

experimental studies using primary human hepatocytes

exposed to comparably high bile acid concentrations, the

induction of cytolytic cell destruction has been demon-

strated [9]. In most patients with cholestatic liver disease,

however, such high bile acid concentrations are not

observed [3]. Therefore the aim of this study was to

evaluate hepatocellular damage induced by low bile acid

concentrations of 50±100 mM GCDCA, representing the


Figure 1 TUNEL (in situ dUTP nick-end labelling technique)-

stained primary human hepatocytes after 4 h of incubation with

50 mM and 100 mM GCDCA. Treatment with 50 mM GCDCA

was followed by cytoplasmic vacuolization (a) (´ 800). Apoptotic

cell damage was indicated by positive nick-end labelled nuclear

chromatin that became marginated, and by hepatocellular

shrinkage (b) (´ 800). Cells that were co-incubated with 50 mM

GCDCA and 50 mM TUDCA showed predominantly cytoplas-

mic lesions (c) (´ 800). Following treatment with 100 mM

GCDCA, hepatocellular damage was characterized by entirely

TUNEL-positive nuclei and by cytoplasmic fragmentation (d)

(´ 800).



range of moderately elevated bile acid concentrations

which might characterize the early states of chronic chole-

static liver diseases such as primary biliary cirrhosis and

primary sclerosing cholangitis.

After exposure to 100 mM GCDCA for 4 h, the induc-

tion of cytolytic cell damage in primary cultured human

hepatocytes was indicated by signi®cantly increased AST

release and by morphological signs of membrane destruc-

tion. Under these experimental conditions, co-incubation

with equimolar concentrations of TUDCA had no effect.

This differs from studies with prolonged exposure to

hydrophobic bile acids demonstrating that cytolytic cell

Figure 2 Electron microscopy of primary human hepatocytes

after 4 h of incubation with 50 mM GCDCA revealed the forma-

tion of extensive lipid vacuoles (a) (´ 5500). Apoptotic cell death

was indicated by hepatocellular shrinkage, by the aggregation of

intracellular organelles and by the condensation and margination

of nuclear chromatin (b, upper cell) (´ 5000). These alterations

were reduced by co-incubation with equimolar concentrations of

TUDCA (c) (´ 5000). Treatment with 100 mM GCDCA induced

apoptotic nuclei but also extensive cytolytic cell destruction (d)

(´ 13 500). L, lipid vacuoles; M, mitochondrion; rER, rough

endoplasmic reticulum.

208 C. Benz et al.

destruction was signi®cantly reduced by co-incubation

with ursodeoxycholic acid [9]. Therefore short incubation

may not be suf®cient to evaluate an effect of TUDCA on

bile acid-induced cytolysis in primary human hepatocytes.

To evaluate the role of apoptotic cell death induced by

bile acids, cytolytic cell destruction was excluded by using

low bile acid concentrations of 50 mM. Under these condi-

tions, hepatocellular enzyme release was not signi®cantly

altered and light microscopy showed preserved cell mem-

branes. However, bile acid-induced cell damage was indi-

cated by morphological alterations and by biochemical

evidence of nuclear DNA fragmentation. As demonstrated

by the TUNEL reaction and by electron microscopy, bile

acid-induced apoptosis was indicated by the condensation

and margination of nuclear chromatin, which is con-

sidered to represent the hallmark of apoptotic cell death

[19]. In addition, the compaction of the cytoplasm and the

aggregation of intracellular organelles corresponded to

characteristic signs of apoptosis [20,21]. However, ultra-

structural evaluation of human hepatocytes failed to

demonstrate the formation of membrane-bound apoptotic

bodies indicating end-stage apoptosis. This might be due to

the fact that apoptosis represents an active energy-depen-

dent process, which may be disturbed in energy-depleted

cultured primary human hepatocytes after prolonged pre-

paration procedures. Taken together, these morphological

studies demonstrate for the ®rst time characteristic signs of

apoptosis in primary cultures of human hepatocytes after

short exposure to bile acids. The variable rate of TUNEL-

positive cells in different sections of TUNEL-stained slides

hampered reliable statistical evaluation. Therefore a highly

sensitive ELISA method was used to measure apoptotic

DNA cleavage. In contrast to our results in primary rat

hepatocytes, oligonucleosomal DNA fragmentation was

not enhanced by GCDCA concentrations higher than

50 mM. This might be due to the fact that higher

GCDCA concentrations are not followed by increased

apoptotic cell damage because they rapidly induce cytolytic

cell destruction. Co-incubation with equimolar concentra-

tions of TUDCA was followed by signi®cantly reduced

oligonucleosomal DNA cleavage, which approached the

level of control cells. These ®ndings suggest a bene®cial

role of TUDCA in reducing bile acid-induced liver cell

damage at low bile acid concentrations.

In rat hepatocytes, the mechanism of bile acid-induced

apoptosis has been partially elucidated, but the initial

apoptosis-triggering event is unknown. Activation of protein

kinase C seems to occur early and may lead to the activation

of a cascade of proteases, including stepwise activation of

cathepsin D and B, which ®nally may induce apoptotic

DNA fragmentation by the activation of magnesium-

dependent endonucleases [22,23]. However, other mecha-

nisms, such as the generation of oxygen radicals leading to

increased lipid peroxidation, may also contribute to hepa-

tocellular apoptosis [24]. It is not known whether apoptotic

cell death, observed in mouse liver after exposure to low

concentrations of several hepatotoxic agents [25±27],

might be due to toxin-induced Fas receptor overexpression.

In human hepatocytes, the Fas receptor has been reported

to be expressed constitutively at low levels [28]. In con-

trast, in livers of patients suffering from viral hepatitis or

fulminant liver failure, Fas receptor expression was found

to be increased [14] and it was suggested that, under

these conditions, hepatocytes might be killed by soluble

systemic Fas ligand or by cell surface Fas ligand on T

cells [29]. Moreover, it has been suggested that in Hep

G2 cells, bile acid-induced apoptosis is induced by activa-

tion of the Fas system [30]. In this study, as demonstrated

by quantitative evaluation, exposure of primary human

hepatocytes to low GCDCA concentrations was not

followed by signi®cantly increased Fas receptor expres-

sion. Therefore it can be concluded that the triggering


Figure 3 Nuclear DNA fragmentation after incubation of 106

cells for 4 h with medium (B), 50 mM GCDCA (X) and 50 mM

GCDCA �50 mM TUDCA (O) measured by the use of a cell

death detection ELISA. Results are expressed as relative ratios

(apoptosis index) of absorbance of sample cells to absorbance of

control cells (mean SD, n�8). *A signi®cant reduction of the

GCDCA-induced nuclear DNA fragmentation by co-incubation

with TUDCA (P<0´05).

Figure 4 Hepatocellular Fas receptor expression after incuba-

tion of 106 cells for 4 h with medium, 50 mM GCDCA, 50 mM

GCDCA �50 mM TUDCA, 100 mM GCDCA and 100 mM

GCDCA � TUDCA 100 mM measured by the use of a Fas anti-

gen ELISA. Quanti®cation of Fas receptor showed no signi®cant

differences between the experimental groups.



event in bile acid-induced apoptosis is not mediated by

enhanced hepatocellular Fas receptor expression.

In primary human hepatocytes the induction of apopto-

tic cell death due to low concentrations of bile acids has

been demonstrated by morphological evaluation and by

quanti®cation of oligonucleosomal DNA cleavage. The

reduction of nuclear DNA fragmentation by co-incubation

with TUDCA was comparable to our previously described

results in primary rat hepatocytes. These ®ndings in pri-

mary human hepatocytes suggest that, in moderately severe

cholestasis with slightly elevated bile acid concentrations,

bile acid-induced apoptosis represents an important

mechanism of cell damage that, at least partially, may be

prevented by ursodeoxycholic acid and its conjugates.

Acknowledgements

We thank Petra KloÈters-Plachky and Annette Stradtmann

for their excellent technical assistance.

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effect of tauroursodeoxycholic acid on bile acid-induced apoptosis in primary human hepatocytes

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