1 Departments of Internal Medicine and 2 Medical Physiology, 3 Division of Research and Education, Scott & White Hospital and The Texas A&M University System Health Science Center, College of Medicine and 4 Central Texas Veterans Health Care System, Temple, Texas 76504; 5 Third Department of Internal Medicine, Tohoku University School of Medicine, Aobaku, Sendai, Japan 980-8574; and 6 Division of Gastroenterology, University of Rome, La Sapienza, Rome, Italy 00100
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ABSTRACT |
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Cholangiocyte proliferation and loss through apoptosis
occur in cholestatic liver diseases. Our aim was to determine the
mechanisms of apoptosis in an animal model of ductal
hyperplasia. Rats were fed -naphthylisothiocyanate (ANIT) for 2 wk
and subsequently fed normal chow for 1, 2, and 4 wk. Proliferation was
assessed in sections by morphometry and in small and large
cholangiocytes by proliferating cellular nuclear antigen immunoblots
and measurement of cAMP levels. Apoptosis and reactive oxygen
species (ROS) levels were also assessed. ANIT feeding increased small
and large cholangiocyte proliferation and apoptosis. Cessation
of ANIT feeding was associated with decreased proliferation and a
further increase in apoptosis in small and large
cholangiocytes. Cholangiocytes from ANIT-fed rats or exposed to ANIT in
vitro showed increased apoptosis and ROS generation.
ANIT-induced duct injury results in enhanced proliferation and
apoptosis in small and large cholangiocytes. The mechanism of
ANIT-induced apoptosis may be due to ROS generation induced directly by ANIT. Our model has implications for understanding the
pathophysiology of cholangiopathies (characterized by the coexistence
of cholangiocyte apoptosis and proliferation).
cyclic adenosine 3',5'-monophosphate; intrahepatic biliary epithelium; DNA replication; reactive oxygen species
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INTRODUCTION |
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IN THE NORMAL STATE,
CHOLANGIOCYTES are mitotically quiescent (3, 32);
however, they proliferate in response to injury/toxins, including
bile duct ligation (BDL) (4-6), CCl4
(33, 35), and -naphthylisothiocyanate (ANIT) feeding
(5), resulting in ductal hyperplasia or damage
(3-6, 33, 35). A number of studies (25,
26, 38) have shown that ANIT treatment alters hepatic
glutathione levels.
In human liver diseases characterized by chronic cholestasis, cholangiocytes markedly proliferate (6, 14-16, 44). Proliferation may be triggered by local inflammation (39), increased ductal pressure (47), increased bile acids (2), or alteration of portal nerve function or hormone signaling (31). Cholangiocyte loss, due to apoptosis leading to ductopenia, is also observed in these diseases (11, 12, 27, 31, 33, 35, 39). Apoptosis may stimulate cholangiocyte proliferation to compensate for loss of ductal mass and function (33, 35). For instance, acute CCl4 administration induces cholangiocyte loss through apoptosis, which subsequently results in compensatory cholangiocyte proliferation (33, 35). The CCl4 model of cholangiocyte apoptosis is dissimilar to human liver diseases in which apoptosis and proliferation coexist (14, 15). The cellular mechanisms resulting in cholangiocyte apoptosis in animal models and human liver diseases remain obscure.
The intrahepatic biliary tree is heterogeneous regarding morphology and
secretory, proliferative, and apoptotic activities (1, 3, 7,
8, 28, 33, 35). Specifically, small ducts (15 µm in
diameter) are lined by small cholangiocytes (8 µm in size), whereas
larger ducts (>15 µm in diameter) are lined by larger cholangiocytes
(13 µm in size) (1, 7). Large ducts are the major
anatomic sites of secretin-stimulated ductal secretion (1, 7,
28), whereas small ducts do not respond to secretin (1, 7,
28). Following BDL, cholangiocyte proliferation is restricted to
large cholangiocytes (3, 33). In contrast, after 70%
hepatectomy (34) or CCl4-induced duct damage
(33, 35), small and large ducts proliferate, contributing
to the regrowth of the biliary tree. The function of small
cholangiocytes remains undefined due to the unavailability of
experimental models to study the pathophysiology of small cholangiocytes.
One of the mechanisms thought to trigger apoptosis is the accumulation of reactive oxygen species (ROS) in cells (37). The increase in intracellular ROS levels correlates with cell death caused by a variety of proapoptotic stimuli, including drugs (30). Antioxidants protect cells from apoptosis, and H2O2 induces apoptosis in a variety of cells (13). Although a role for ROS in transducing apoptotic signals from the extracellular environment has been suggested, recent studies suggest that ROS may act downstream of the caspase system (48).
The aim of this study was to develop a model of coexisting cholangiocyte proliferation and apoptosis and to determine if ROS plays a role in cholangiocyte apoptosis. We first fed normal rats 0.1% ANIT or control diet for 2 wk and subsequently fed them normal chow for 1, 2, and 4 wk. At the selected feeding times, we measured proliferation and apoptosis in isolated small and large cholangiocytes. To evaluate the mechanisms by which small and large cholangiocytes undergo ANIT-induced apoptosis, we measured both in vivo and in vitro ROS in small and large cholangiocytes.
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MATERIALS AND METHODS |
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Animal model. Male Sprague-Dawley rats (150-175 g) were purchased from Charles River (Wilmington, MA). The animals were kept in a temperature-controlled environment (22°C) with a 12:12-h light/dark cycle. The studies were conducted in 1) rats fed 0.1% ANIT or control diet (AIN 76) for 2 wk and 2) rats first fed 0.1% ANIT for 2 wk and subsequently fed normal chow for 1, 2, and 4 wk. Before each procedure, animals were anesthetized with sodium pentobarbital (50 mg/kg ip). In all of the animals used, we measured wet liver weight, body weight, and wet liver weight-to-body weight ratio.
Materials. Reagents were purchased from Sigma Chemical (St. Louis, MO) unless otherwise indicated. Rat chow was prepared by Dyets (Bethlehem, PA). Porcine secretin was purchased from Peninsula Laboratories (Belmont, CA). RIA kits for the determination of intracellular cAMP levels were purchased from Amersham (Arlington Heights, IL). The mouse anti-cytokeratin 19 (CK-19) antibody was purchased from Amersham. The monoclonal mouse antibody against proliferating cellular nuclear antigen (PCNA) was purchased from DAKO (Kyoto, Japan). A biotinylated antibody reacting with annexin-V was purchased from Boehringer Mannheim (Mannheim, Germany).
Purification of small and large cholangiocytes and ducts.
Pure small and large cholangiocytes [by -glutamyltranspeptidase
histochemistry (46)] were purified by immunoaffinity bead separation (7, 8, 35) by using a mouse monoclonal oval cell-2 antibody (IgM; kindly provided by Dr. R. Faris, Brown
University, Providence, RI) against an unidentified membrane antigen
expressed by all rat intrahepatic cholangiocytes (23).
Cell viability was ~97%. Isolated small (<15 µm in diameter) and
large (>15 µm in diameter) intrahepatic bile duct units (IBDU) were
obtained from small and large ducts by employing liver perfusion with
collagenase, manual stripping of portal structures, and recovery of
dissociated IBDU with a pipette under a dissecting microscope as
previously described by us and others (1, 36, 43). We
performed in situ morphometric analysis in small and large purified
IBDU (1) and found that, as observed in normal rats
(1, 36, 43), small cholangiocytes line small ducts and
large cholangiocytes line large ducts in ANIT-fed rats.
Effect of ANIT feeding on proliferation and apoptosis
proliferation.
Cholangiocyte proliferative capacity was evaluated in liver sections by
quantitative localization of PCNA in small and large intrahepatic bile
ducts. PCNA is a nonhistone nuclear protein that plays an important
role in DNA replication and cellular proliferation by interacting with
DNA polymerase- (29). In situ immunohistochemistry for
PCNA was performed as follows. After blocking of endogenous peroxidase
activity by methanol-peroxidase solution, nonspecific binding was
blocked by incubation with normal goat serum for 30 min at room
temperature. Paraffin-embedded sections (5 µm thick) were incubated
with anti-PCNA antibody (clone PC-10; Oncogene Research Products,
Cambridge, MA) at 4°C overnight. After several washes with cold PBS,
biotin-labeled secondary antibody at a dilution of 1:100 (DAKO) was
added for 1 h at room temperature. To detect this reaction, a
peroxidase-labeled avidin/biotin complex with diaminobenzidine (Dojin
Chemical, Kumamoto, Japan) was used as a substrate. Over 100 small and
large cholangiocytes were counted in three different fields for each
group of animals. Sections were counterstained with hematoxylin and
examined with a microscope (Olympus BX 40; Olympus Optical, Tokyo, Japan).
Apoptosis. Small and large cholangiocytes purified from control or ANIT-fed rats and rats fed ANIT followed by normal chow for 1, 2, and 4 wk were analyzed for apoptosis by annexin-V staining [a specific marker of early-stage apoptosis (20)] as previously described by us (31). Briefly, purified small and large cholangiocytes (1 × 106) were incubated for 10 min with a biotinylated antibody reacting with annexin-V 1:50 dilution in HEPES buffer (10 mM HEPES, pH 7.4, 140 mM NaCl, and 5 mM CaCl2) and spun down with a cytospin device (100 g for 5 min) onto glass slides. Cell smears were soaked in methanol/ethanol 1:1 for 90 s and incubated for 1 h at room temperature with streptavidin-horseradish peroxidase conjugate (1:1,000 dilution in HEPES buffer). Following washes with HEPES buffer, cell smears were incubated with diaminobenzidine (1:9 solution in H2O2) solution for 15 min, counterstained for 10 s with hematoxylin solution, and examined by light microscopy with an Olympus BX 40 microscope equipped with a camera. At least 300 cells/slide were counted in a coded fashion.
Does ANIT in vitro induce cholangiocyte apoptosis? We next evaluated in purified pooled cholangiocytes [which are composed of small and large cholangiocytes (3, 7, 8)] from normal rats whether ANIT induces in vitro cholangiocyte apoptosis and if the oxygen scavenger ethyl ester glutathione prevents the increase in cholangiocyte apoptosis due to in vitro ANIT treatment. Purified pooled cholangiocytes from normal rats were treated for 2 h at 37°C with ANIT (1-10 µM), H2O2 (100 µM), or ANIT plus ethyl ester glutathione (2 µM). Subsequently, cells were incubated with 1 µM 4,6-diamidino-2-phenylindole (DAPI) for 10 min at 37°C and transferred to the stage of a Nikon Diaphot microscope equipped with fluorescence and differential interference optics where DAPI fluorescence was visualized with the use of excitation and emission filters of 380 and 460 nm, respectively. Nuclei were visualized from the fluorescent DNA binding dye DAPI (40). Cholangiocytes were considered apoptotic if nuclear invagination, condensation, and fragmentation were observed (40); 300 cells were analyzed for each experimental group. Apoptosis was expressed as total of the cells counted. We used as a positive control normal cholangiocytes treated with beauvericin (10 µM) for 4 h at 37°C (33, 35, 40).
Measurement of ROS.
We next evaluated intracellular ROS concentration in
1) small and large cholangiocytes purified from rats fed
control or ANIT diet and rats fed ANIT followed by normal food for 4 wk
and 2) small and large normal cholangiocytes treated in
vitro at 37°C with ANIT (10 µM for 2 h),
H2O2 (100 µM for 2 h), or ethyl ester glutathione (2 µM for 15 min) + ANIT (10 µM for 2 h). The
H2O2 concentration in cholangiocytes was
measured by employing the ROS indicator dye 2',7'-dichlorofluorescein
(DCF; Molecular Probes, Eugene, OR). DCF is nonionized and membrane
permeant and is thus able to diffuse into cells (24).
Within the cell, the acetate groups are removed by intracellular
esterase, resulting in a polar molecule that is trapped within cells.
DCF fluoresces when it is oxidized by H2O2 or
lipid peroxides (24). The level of DCF fluorescence in
cells is related linearly to the amount of peroxides present within the
cell. DCF stock (103 M) was prepared in DMSO and diluted
to 10
5 M to add to cells. After 30 min of incubation,
excess extracellular DCF was removed by washing and cells were mounted
on the stage of a Nikon fluorescence microscope. The fluorescence was
quantitated by a cooled charge-coupled device camera (Photometerics),
and data were analyzed by employing Isee software (Enovision, Durham, NC). The regions of interest, defined by cell cytoplasm, were identified, and total fluorescence for each cell was quantitated.
Statistical analysis. All data are expressed as means ± SE. The differences between two groups were analyzed by Student's t-test.
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RESULTS |
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Liver weight and liver weight-to-body weight ratio.
The wet liver weight-to-body weight ratio was higher in ANIT-fed rats
compared with controls (Table 1).
Following cessation of ANIT feeding, the wet liver weight-to-body
weight ratio decreased but remained higher than in control-fed rats
(Table 1).
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Proliferation.
Few PCNA-positive small and large cholangiocytes were observed in
control sections (Fig. 1). The number of
PCNA-positive small and large cholangiocytes significantly
(P < 0.05) increased following ANIT feeding (Fig. 1).
At cessation of ANIT feeding, there was a gradual decrease in the
number of PCNA-positive cholangiocytes, which, however, remained higher
than control values (Fig. 1).
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Apoptosis.
We evaluated whether alterations in cholangiocyte proliferation
are associated with temporal and reciprocal changes in cholangiocyte apoptosis. We found that ANIT-induced increases in
proliferative capacity of small and large cholangiocytes were
associated with apoptosis of these cells (Fig.
5). Regression of cholangiocyte proliferation (at cessation of ANIT feeding) was associated with a
further increase of apoptosis of small and large cholangiocytes (Fig. 5). However, at 4 wk after cessation of ANIT feeding,
apoptotic activity, although lowered, remained higher than in
control cholangiocytes (Fig. 5). The direct effects of ANIT on
cholangiocyte apoptosis were first determined in vitro in
pooled cholangiocytes, which include small and large cholangiocytes
(3, 7, 8). Dose-dependent studies showed that 1, 2, and 10 µM ANIT induced apoptosis in 0.8 ± 0.2, 2.1 ± 0.6, and 6.5 ± 1.1% of cholangiocytes, respectively. Since the
10-µM concentration induced apoptosis to a similar degree to
that observed in vivo, we employed this concentration for the studies.
ANIT increased cholangiocyte apoptosis; however, pretreatment of cholangiocytes with ethyl ester glutathione reduced ANIT-induced apoptosis (Fig. 6).
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Measurement of ROS.
Small and large cholangiocytes were isolated from control, ANIT-fed
rats and ANIT-fed rats subsequently fed normal food for 4 wk. DCF
fluorescence, which is proportional to intracellular ROS concentration,
was higher in ANIT-fed rats, as shown by the shift of the fluorescence
histogram to the right (higher fluorescence) in both small and large
cholangiocytes (Fig. 7). In rats fed ANIT and then control diet, ROS generation decreased but was higher than in
control rats (Fig. 7).
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DISCUSSION |
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These studies show that ANIT administration to rats induces coexisting cholangiocyte apoptosis and proliferation. During ANIT feeding, both small and large cholangiocytes (lining small and large ducts, respectively) proliferate and undergo apoptosis. During ANIT feeding, proliferation is the predominant event, since we observed an increase in duct mass. After cessation of ANIT feeding, apoptosis became the predominant event, with a progressive decline in proliferation but increase in cholangiocyte apoptosis, resulting in a reduction of duct mass. In vitro, we found that ANIT directly increased apoptosis and ROS levels in cholangiocytes and inhibition of ROS prevented ANIT-induced apoptosis. In vivo, ROS generation in cholangiocytes from ANIT-fed rats was greater than control both during and after ANIT feeding. These studies show that ANIT-induced cholangiopathy is similar to human cholestatic liver diseases, with the presence of apoptosis coexisting with proliferation (14, 15). Also, the overall duct mass resulted from a balance between proliferation and apoptosis. Finally, these studies establish a role for ROS in a drug-induced model of duct injury for both the induction of apoptosis and the persistence of apoptosis after drug withdrawal.
Small or large ducts may be exclusively involved in human cholestatic liver disease (3, 6, 33, 35). In primary biliary cirrhosis, larger intrahepatic bile ducts show apoptosis, whereas in drug-induced ductopenia, smaller ducts have apoptotic cholangiocytes (14-16). Similarly, in animal models, proliferation or apoptosis is restricted to certain sizes of ducts (3, 31, 33, 35). BDL induces proliferation only in large bile ducts (3, 33). CCl4 induces de novo proliferation only in small cholangiocytes (33, 35), whereas partial hepatectomy causes proliferation of both small and large cholangiocytes (34). CCl4 induces cholangiocyte apoptosis like ANIT; however, apoptosis is restricted to large ducts (33, 35). Thus the ANIT model is unique among animal models of cholangiopathies, with both coexisting apoptosis and proliferation present in small and large ducts.
The effect of ANIT on cholangiocyte apoptosis was persistent even 4 wk after cessation of treatment. This finding contrasts with other models of ductal hyperplasia (BDL, partial hepatectomy, and acute CCl4 treatment), in which cholangiocyte proliferation is transient (32-35) or completely normalizes with removal of injury (10). Although cholangiocyte apoptosis may completely disappear after 4 wk, this represents the first described model of persistent cholangiocyte apoptosis. Also, the model has relevance to drug-induced ductopenic syndromes, in which, even after removal of the drug, cholestasis, once acquired, remains persistent for periods of up to 1 yr (16).
The apoptotic process consists of an activation and an execution phase (45). Previous studies (22) have suggested that ROS may be a key element in the activation of apoptosis in many cell types, including cholangiocytes (42). ROS levels in cells correlate with the presence of cell death (22). Antioxidants protect cells from apoptosis, and experimentally increased ROS levels induce apoptosis (22). Increased Bcl-2 expression is protective against apoptosis, and Bcl-2 may at least partially function as an intracellular antioxidant (19). Other mechanisms for ROS activation of apoptosis may involve activation of transcriptional factors or kinases (42). We demonstrated that ANIT or H2O2 in vitro induces ROS formation in small and large cholangiocytes. The ANIT-induced increase in ROS and apoptosis in purified cholangiocytes is prevented by pretreatment with the antioxidant ethyl ester glutathione. The data show that ANIT can directly induce apoptosis, and we propose that the initial increase in apoptosis during ANIT feeding is due at least in part to the direct effects of ANIT on cholangiocyte ROS production. Since ANIT has been shown to alter hepatic glutathione levels (25, 26, 38), low cholangiocyte glutathione may predispose cholangiocytes to ROS generation and ROS-mediated apoptosis in this model. In vivo, ROS is increased in both small and large cholangiocytes while rats are on the ANIT diet. Subsequent to removal of ANIT from the diet, cholangiocyte ROS levels decrease but remain at values higher than controls. The persistence of high ROS production after ANIT removal may be due to persistence of portal inflammation that occurs in the ANIT model, with cytokine release from periductular inflammatory cells inducing ROS production. The observation of a persistent ROS increase after ANIT removal is potentially important, since it suggests that antioxidant drugs could be therapeutic in the recovery from a prolonged phase of drug-induced duct injury (6, 44) or in chronic liver diseases with persistent ductopenia (6, 44).
Cholangiocyte proliferation was evaluated by several complementary independent methods. Similar to our previous studies (2, 3, 18, 32, 35) in rats with BDL, partial hepatectomy, and fed bile acids, we have found increased cholangiocyte PCNA expression, increased number of PCNA-positive cholangiocytes and CK-19-positive ducts, and increased secretin-stimulated cAMP levels in proliferating cholangiocytes. Our data are consistent with previous studies demonstrating that ANIT feeding induces cholangiocyte proliferation (5, 10) and other studies showing that (following ANIT withdrawal) cholangiocytes undergo apoptosis (10). However, the previous studies (5, 10) did not show that apoptosis coexists with proliferation, determine the size of intrahepatic bile ducts affected by ANIT, or demonstrate the involvement of ROS in cholangiocyte apoptosis as we did in this study.
In summary, these studies demonstrate the relationship between cholangiocyte proliferation and apoptosis as they determine bile duct structure in a drug-induced bile duct injury model. The studies may be important to better understand the course of cholestatic liver diseases characterized by the coexistence of cholangiocyte damage and proliferation, the latter representing a mechanism of bile duct repair, which determines the outcome of the disease (14, 15). These studies for the first time implicate increased ROS generation in the initiation and persistence of bile duct injury in an animal model, which could also happen in human cholangiopathies. The potential for cytoprotective action through antioxidant therapy in cholangiopathies should be explored. Finally, the study underscores the importance of evaluating the zonal nature of different forms of bile duct injury (6).
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ACKNOWLEDGEMENTS |
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This work was supported by a grant award to G. LeSage and G. Alpini from Scott & White Hospital and The Texas A&M University System, by National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) Grant DK-54208 to G. LeSage, and by a Veterans Affairs Merit Award and NIDDK Grant DK-58411 to G. Alpini.
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FOOTNOTES |
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Address for reprint requests and other correspondence: G. Alpini, Associate Professor, Internal Medicine and Medical Physiology, The Texas A&M Univ. System HSC, COM and Central Texas Veterans HSC, MRB, 702 South West H.K. Dodgen Loop Temple, TX 76504 (E-mail: falpini{at}mailbox.sw.org).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 10 January 2001; accepted in final form 8 March 2001.
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