Effects of Ursodeoxycholic Acid and Chenodeoxycholic Acid on Human Hepatocytes in Primary Culture S O P H I E HILLAIRE, 1 FRANCOIS BALLET, 2 D O M I N I Q U E FRANCO, 3 K E N N E T H
D. R. SETCHELL, 4 AND R A O U L P O U P O N 1
Bile acids are amphiphilic molecules, the most hydrophobic of which have detergent properties and are cytotoxic. They are synthesized from cholesterol by hepatocytes and are eliminated in the bile by the action of specific canalicular transporters. In chronic cholestatic diseases, bile acid concentrations increase in both the circulation and the liver because of a reduction in their canalicular excretion, together with ileal absorption of the fraction eliminated in the digestive tract. 1 The toxicity of bile acids might thus partly account for the observed liver damage. In chronic cholestatic liver diseases, particularly prim a r y biliary cirrhosis, ursodeoxycholic acid (UDCA) t r e a t m e n t improves the clinical, biological, and histological manifestations,2 possibly through a reduction in the active ileal absorption of endogenous bile acids. 3'4 However, this would not account for the preventive effect of UDCA on the cholestasis induced by lithocholic acid in the isolated perfused rat liver, ~'6 nor the reduction in rat hepatocyte necrosis caused by chenodeoxycholic acid (CDCA) after intravenous injection of UDCA. 7's This latter effect could be attributable either to a decrease in the hepatocyte concentration of toxic bile acids or to a direct cytoprotective effect of UDCA. The aim of this study was to assess changes in the viability and function of primary cultured h u m a n hepatocytes induced by CDCA, UDCA, and tauro-UDCA (TUDCA), and to test the potential direct protective effect of UDCA and its conjugated form against the cytotoxic action of CDCA.
Hepatic bile acid c o n c e n t r a t i o n s are elevated in chronic cholestasis b e c a u s e of r e d u c e d canalicular excretion and active ileal absorption of the fraction elimi. nated in the gut. U r s o d e o x y c h o l i c acid (UDCA) reduces the intestinal absorption of e n d o g e n o u s bile acids, thereby diminishing the concentrations to w h i c h liver cells are exposed. In the isolated perfused liver (in w h i c h vectorial bile acid transport is maintained), UDCA reduces the cytotoxic and cholestatic effects of endogen o u s bile acids. As a result, it has b e e n suggested that UDCA or o n e of its conjugates could have a direct protective effect on h e p a t o c y t e structure and function. We therefore studied the effects of c h e n o d e o x y c h o l i c acid (CDCA) and t a u r o u r s o d e o x y c h o l i c acid (TUDCA) alone and in c o m b i n a t i o n o n the viability and certain functions of h u m a n h e p a t o c y t e s in primary culture. TUDCA did not affect intracellular concentrations of CDCA w h e n added concomitantly. In other experiments, CDCA (100 to 500 ftmol/L) i n d u c e d concentration-dependent increases in lactate d e h y d r o g e n a s e (LDH) leakage and decreases in cellular protein synthesis and albumin secretion. Neither TUDCA nor UDCA had similar effects at the same concentrations, nor did t h e y h a v e a protective effect w h e n added c o n c o m i t a n t l y w i t h CDCA at equimolar or twice-equimolar concentrations. These results suggest that UDCA has no direct cytoprotective effect w h e n the bile acid c o n c e n t r a t i o n s to w h i c h h u m a n hepatocytes are e x p o s e d are u n c h a n g e d . They also suggest that the hepatoprotective effect of UDCA in cholestatic h u m a n liver diseases and in the isolated perfused liver loaded w i t h h y d r o p h o b i c bile acids occurs through its effect on intestinal and h e p a t o c y t e transport systems. (HEPATOLOGY 1995;22:82-87.)
MATERIALS AND METHODS
Hepatocyte Isolation. Human liver fragments were obtained with informed consent from patients undergoing hepatectomy because of liver tumors. Hepatocytes were isolated by means of two-step collagenase perfusion as described elsewhere. 9 Cell viability, assessed by refringence under phasecontrast microscopy, was better than 80%. Hepatoeyte C u l t u r e . Hepatocytes were incubated at a density of 1.5 × 105 cm2 in 10 × 35 mm2 uncoated plastic petri dishes (Nunc, Denmark), in 1.5 mL L15 medium containing fetal calf serum (10%), NaHCQ (26 mmol/L), bovine albumin (0.2%), penicillin (10 IU/mL), streptomycin (10 #g/mL), glutamine (2 mmol/L), and insulin (100 IU/L), at 37°C in a humidified atmosphere of 5% COJ95% air in Stericnlt Forma Scientific (Bioblock). Once the cells had attached (6 to 12 hours), the medium was replaced with serum-free medium supplemented with 10-8 mol/L hydrocortisone hemisuccinate. The medium was renewed daily. The hepatocytes were tested
Abbreviations: UDCA, ursodeoxycholic acid; CDCA, chenodeoxycholic acid; TUDCA, tauroursodeoxycholic acid; LDH, lactate dehydrogenase. From the 1Uuit~ d'H~patologie, HSpital Saint-Antoine, Paris, France; the 2Drug Safety Department, Rh6ne-Poulenc Rorer, Vitry-Alforville, France; 3Service de Chirurgie G~n~rale, HSpital Antoine B~cl~re, Clamart, France; and 4Children's Hospital Medical Center, Clinical Mass Spectrometry, Cincinnati, OH. Received September 28, 1994; accepted January 27, 1995. Address reprint requests to: Raoul Poupon, MD, Service d'h~pato-gastroent~rologie, HSpital Saint-Antoine, 184 rue du faubourg Saint-Antoine, 75012 Paris, France. Copyright © 1995 by the American Association for the Study of Liver Diseases. 0270-9139/95/2201-001253.00/0 QO
HEPATOLOGYVol. 22, No. 1, 1995 after 2 days of culture, by treatment with L15 medium containing the following bile acids: (1) CDCA 100 to 500 #mol/ L; (2) UDCA 100 to 500 #mo]/L; (3) TUDCA 100 to 500 #tool/ L; (4) CDCA + UDCA or TUDCA at various concentrations. Control hepatocytes were incubated in L15 medium alone. Bile acid concentrations were determined using an enzymatic method (3-a-hydroxydehydrogenase assay, Enzabile, Nycorned, Oslo, Norway). Cell Harvest. At the end of the incubation period, the supernatant was drawn off into cryotubes, which were immediately placed in liquid carbogen and stored at -80°C until the assay of enzyme activities and albumin content. The cell monolayers were scraped with a rubber policeman after two washes with 1.5 mL phosphate-buffered saline and collected in 1 mL of the same buffer. The pellet was homogenized, rapidly cooled in liquid carbogen, and stored at -80°C. Bile Avid Analysis. Bile acid concentrations were determined in the culture media using a combination of high-pressure liquid chromatography and gas chromatography-mass spectrometry techniques. The culture media (2 mL) was separated from the cells, and nordeoxycholic acid (5 #g) was added as an internal standard for quantifying the concentrations of unconjugated bile acids. The sample was diluted with 4 vol 0.1 mol/L sodium hydroxide, heated to 64°C, and the bile acids were extracted by solid-phase absorption using cartridges of octadecylsilane-bonded silica exactly as described previously. 1° Unconjugated bile acids were separated from the glycine, taurine, and sulfate conjugated by lipophilic anion exchange chromatography. 11 The unconjugated bile acids were converted to methyl ester-trimethylsilylether derivates and analyzed by gas chromatography and mass spectrometry. 12 The conjugated bile acid fractions were taken to dryness, reconstituted in mobile phase, and analyzed by reverse-phase HPLC essentially as described by Rossi et al. 13 Protein Synthesis Assay. Total protein synthesis was determined by labeling hepatocytes with [~4C]-valine (285 mCi/ mmol, 0.1 #Ci/dish) for 24 hours in culture medium containing 1.7 mmol/L cold valine. The medium was removed at the end of the incubation period. The cells were washed three times with 2 mL phosphate-buffered saline at 4°C, precipitated with 1 mL 5% perchloric acid, scraped with a rubber policeman, and centrifuged for 30 minutes at 4,500 rpm. The supernatant was removed, and the pellet was hydrolyzed in 1 mL 1 mol/L NaOH for 1 hour at 37°C. An aliquot was used for protein determination, and [14C]-valine incorporation was determined by means of scintillation counting. The results are expressed as the percentage inhibition of protein synthesis in the presence of bile acids relative to untreated controls. Albumin Secretion. The concentration of h u m a n albumin in the hepatocyte culture supernatants was determined using a radioimmunoassay (hAlb. kit, Biointernational, Gif-surYvette, France), which shows no cross-reaction with bovine albumin. LDH Leakage. Lactate dehydrogenase (LDH) leakage, a marker of cytotoxicity, was calculated as follows: LDH activity in the supernatant/(LDH activity in the supernatant + LDH activity in the cell pellet). LDH was assayed by monitoring reduced form of nicotinamide-adenine dinucleotide consumption during the conversion of pyruvate to lactate. The rate at which absorbance at 340 nm diminished was monitored at 25°C for 3 minutes using a Beckman spectrophotometer. 14 Intracellular Accumulation o f CDCA. Intracellular CDCA accumulation was measured by incubating hepatocytes with
HILLAIRE ET AL 83 [14C]-CDCA (50 mCi/mmol, 0.75 #Ci/well) and various amounts of cold CDCA alone or in combination with UDCA or TUDCA. After 24 hours' incubation, the dishes were placed on ice, and the medium was aspirated. The cells were washed three times with 3 mL cold phosphate-buffered saline, then hydrolyzed with 1 mL normal NaOH at 37°C for 1 hour. An aliquot was taken for protein determination. Radioactivity was measured by means of liquid scintillation counting. Intracellular CDCA accumulation was expressed as nmol CDCA/mL cell protein. The adsorption of labeled CDCA at 0°C was considered to represent nonspecific binding and was subtracted from the values obtained at 37°C. Protein Assay. The protein content of cell homogenates was determined using Bradford's method, is Cell protein hydrolyzed with NaOH was determined using Lowry et al's method, 1~ using bovine albumin as standard. Sources. Collagenase was obtained from Boehringer Corp (Meylan, France). Leibovitz-L15 culture medium, Hank's salt, penicillin, streptomycin, glutamine, fetal calf serum, and phosphate-buffered saline were from Gibco (Cergy Pontoise, France). Hydrocortisone was from Roussel (Paris, France), and insulin was from Novo Industrie Pharmaceutique (Paris, France). The bile acids (sodium salts) were from Calbiochem (San Diego, CA) and were more than 95% pure. Bovine albumin, HEPES (N-hydroxyethipiperazine-N'-2-ethane sulfonic acid), and reduced form of nicotinamide-adenine dinucleotide phosphate were from Sigma (St. Louis, MO). NaC1, KC1, and CaC12 were from Prolabo (Paris, France). [14C]-valine was provided by Amersham (Les Ulis, France), and [14C]-CDCA was from NEN division (Dupont de Nemours, Germany). All other chemicals were of reagent grade. Statistical Analysis. Results were obtained from at least five experiments and are presented as mean +_ SD. Data were analyzed using Student's t-test. RESULTS
B i o t r a n s f o r m a t i o n o f Bile A c i d s in H e p a t o c y t e Culture. Bile acid c o n c e n t r a t i o n s in t h e s u p e r n a t a n t of t h e cultures determined by both gas chromatography-mass s p e c t r o m e t r y a n d h i g h - p r e s s u r e liquid c h r o m a t o g r a p h y t e c h n i q u e s , a f t e r i d a y of i n c u b a t i o n o f h e p a t o c y t e s w i t h different bile acids, i n d i c a t e d significant b i o t r a n s f o r m a t i o n h a d o c c u r r e d w i t h t h e u n c o n j u g a t e d species. F i g u r e I c o m p a r e s t h e h i g h - p r e s s u r e liquid c h r o m a t o g r a p h y profiles of t h e c u l t u r e m e d i a f r o m t h e control (no bile acid i n c u b a t e d ) a n d t h e h u m a n h e p a t o c y t e p r e p a r a t i o n s a f t e r t h e i n c u b a t i o n w i t h CDCA, U D C A , a n d T U D C A . H i g h - p r e s s u r e liquid c h r o m a t o g r a p h y a n a l y sis s h o w e d t h a t u n c o n j u g a t e d C D C A a n d U D C A w e r e e x t e n s i v e l y c o n j u g a t e d w i t h glycine. C o n v e r s e l y , w h e n T U D C A w a s i n c u b a t e d , it r e m a i n e d l a r g e l y u n c h a n g e d . A s m a l l (9% to 10%) b u t significant p r o p o r t i o n of glycoU D C A w a s identified in t h e m e d i a , w h i c h could not be a c c o u n t e d for b y c o n t a m i n a t i o n of c o m m e r c i a l s u p p l y of T U D C A used. W h e t h e r t h i s reflects h e p a t i c deconjug a t i o n followed b y r e c o n j u g a t i o n is u n c e r t a i n . O t h e r a u t h o r s p r o v i d e d e x p e r i m e n t a l evidence for deconjugation of bile acids b y h e p a t o c y t e s . 17 T h e r e l a t i v e p r o p o r t i o n s of t h e u n c o n j u g a t e d a n d c o n j u g a t e d f o r m s of t h e bile acids in t h e c u l t u r e m e d i a w h e n different c o n c e n t r a t i o n s w e r e i n c u b a t e d for 24 h o u r s a r e s u m m a r i z e d in Fig. 2. A t l o w e r c o n c e n t r a -
84 HILLAIREET AL
Control Hepatocytes [ (no bile acid incubated)
Incubatedwith ] ursodeoxycholicacid
_ _ A
Incubatedwith I chenodeoxycholicacid
Incubated with i tauroursodeoxycholicacid Glyco-UDCA D/
FIG. 1. Reverse-phaseHPLC chromatograms of conjugated bile acids in the culture media of human hepatecytes incubated 24 hours with no bile acid (control) and 100 #mol/Lchenodeoxycholic,ursodeoxycholic,and tauroursodeoxycholicacids.
tions (100 #tool/L), most of the unconjugated bile acids were biotransformed by amidation with glycine, but at high concentrations (250 #mol/L) there was a greater proportion of the unchanged bile acid in the culture media after 24 hours. LDH Leakage. CDCA induced LDH leakage from cultured h u m a n hepatocytes in a concentration-dependent m a n n e r (Fig. 3). The effect was apparent at 250 #mo]/L but was only significantly different from control values at 500 #mol/L. Neither UDCA nor TUDCA in-
 Unconjugated  Glycineconjugated  Taurineconjugated 100 80
~ 60 8
100btM 250~M CDCA
1 0 0 p M 250p_M
duced LDH leakage (Fig. 3), or affected CDCA-induced LDH leakage at concentrations of 100 and 250 #mol/L (Fig. 3). Protein Synthesis. Protein synthesis was evaluated in terms of albumin secretion into the culture medium and the incorporation of labeled valine into cell protein. Both CDCA (Fig. 4) and UDCA (Fig. 5) reduced albumin secretion. The effect was more marked with CDCA t h a n with UDCA and was apparent at a lower concentration (100 #tool/L). In contrast, TUDCA (100 to 500 #tool/L) had no effect on albumin secretion (Fig. 5). Similar results were obtained when protein synthesis was determined in terms of labeled valine incorporation (Fig. 6). TUDCA and UDCA failed to reverse the decrease in albumin secretion induced by 100 #mol/L CDCA (Fig. 7). Intracellular Accumulation of CDCA. H u m a n hepatocytes in primary culture accumulated CDCA, with steady-state being reached within 2 hours. Extracellular CDCA concentrations of 100 and 250 #mol/L gave intracel]ular amounts of 4.0 _+ 0.7 and 13.0 _+ 1.0 nmol/ mg cell protein after 24 hours' incubation (n = 4). Steady-state intracellular CDCA quantity was not modified by 100 #mol/L or 250 #mo]/L TUDCA (Fig. 8). Similar results were obtained with UDCA.
Oi!i 100gM 250gM TUDCA
FIG. 2. Biotransformation of bile acids (CDCA, UDCA, TUDCA) in h u m a n hepatocyte culture after 24 hours of incubation. Bile acid concentrations were determined in the supernatant. Significant bietransformation occurred with both the uncenjugated and the conjugated forms.
The aims of this work were to study the toxic effects of a hydrophobic bile acid (CDCA) on the function and viability of h u m a n hepatocytes, after 24 hours of culture, and to determine whether UDCA or its taurocon-
HEPATOLOGY Vol. 22, No. 1, 1995
HILLAIRE ET AL
IOO CDC^ ~
Bile acids c o n c e n t r a t i o n (~tM)
Bile acids c o n c e n t r a t i o n ( ~ M )
FIG. 3. (A) Effect of bile acids (CDCA, TUDCA, UDCA) on LDH leakage from h u m a n hepatocytes in primary culture after 24 hours' incubation. Only CDCA induced LDH leakage. (B) Effect of CDCA and TUDCA on LDH leakage from h u m a n hepatocytes in primary culture after 24 hours' incubation. TUDCA (100 #tool/L) failed to reverse LDH leakage induced by CDCA at 250 #mol/L and 500 #mol/L. Results were similar for TUDCA concentration of 250 #mol/L. Values are means _+ SD (n = 6). LDH leakage was calculated as LDH activity in culture medium/(LDH activity in culture medium + LDH activity in cells).
jugate reduced these effects. CDCA was chosen because, together with cholic acid, it is the main bile acid in the h u m a n enterohepatic circulation. Is In addition, hepatic concentrations of CDCA in vivo correlate positively with the degree of hepatocyte necrosis.18 We used unconjugated forms of CDCA. Conjugation of bile acids reduced but did not suppress bile acid's cytotoxicity. Indeed, when the concentration of conjugated bile acids increased, the same cytotoxic effects are observed. 19'2° The accumulation of CDCA by hepatocytes was proportional to its concentration in the culture medium. UDCA and TUDCA had no effect on the steady-state intracellular concentration of CDCA. CDCA decreased cell viability measured in terms of LDH leakage in a dose-dependent manner. However, the concentration threshold of this effect was higher than that of the reduction in cell protein synthesis and albumin secretion, which were apparent at 100 pmol/ L. At this concentration, most CDCA was conjugated
to glycine (95%, Fig. 2). Neither UDCA nor TUDCA induced LDH release up to 500 #mol/L. UDCA reduced albumin synthesis, but to a lesser extent and at a higher concentration (250 #mol/L) than CDCA. TUDCA had no biological or toxic effects at the highest concentration tested (500 #mol/L). We did not test higher concentrations of TUDCA, but it was previously shown that TUDCA and glyco UDCA induced LDH release at concentrations higher than 5 mmol/L. I9 The cytotoxicity of CDCA was not affected by UDCA or TUDCA, regardless of the parameter studied, a finding that disagrees with some published data. Leushner et al, 21 studying the cytoprotective effect of UDCA on red cell membranes, found that electron spin resonance values were significantly increased by 20 mmol/L CDCA but unaffected by 20 mmol/L UDCA and TUDCA; the effect of CDCA was reversed by 40 mmol/L UDCA. Similarly, H e u m a n et a122 found that the shortterm toxicity (2 hours) of hydrophobic bile acids w a s reduced in the presence of TUDCA. However, the concentrations used in these two studies were far higher (1 mmo]/L to 2 moFL) than those observed in vivo, even in the most severe cholestatic conditions. In addition, p< 0.05
 2sottM  SO0ttM
%. %., %.
FIG. 4. Effect of CDCA on albumin secretion by h u m a n hepatocytes in primary culture after 24 hours of incubation. CDCA reduced albumin secretion in a dose-dependent manner. Values were significantly different from controls with as little as 1O0 #mol/L CDCA. Values are means _+ SD (n = 6).
FIG. 5. Effect of bile acids (CDCA, UDCA, TUDCA) on albumin secretion by h u m a n hepatocytes after 24 hours of incubation. CDCA and UDCA reduced albumin secretion. The effect was more marked with CDCA and occurred at a lower concentration (100 #mol/L). Values are means + SD (n = 5).
HILLAIRE ET AL
HEPATOLOGY July 1995
it ib ij
 Controls •
 , ~ ply[
FIG. 6. Effect of bile acids on protein synthesis by h u m a n hepatocytes in primary culture after 24 hours' incubation. CDCA and UDCA reduced protein synthesis. The effect was more marked with CDCA and occurred at a lower concentration (100 #mol/L). Values are means _+ SD (n = 5). *P < .05 vs. appropriate control.
FIG. 8. Intracellular CDCA concentration in h u m a n hepatocytes in primary culture. Cellular CDCA content was, respectively, 4 _+0.7 and 13 _+ i nmol/mg cell protein for extracellular concentrations CDCA of 100 and 250 #mol/L. The effect of TUDCA (100 and 250 #mol/L) on CDCA content was measured with an extracellular CDCA concentration of 100 #mol/L. TUDCA did not affect the CDCA concentration when added concomitantly (similar results were obtained with UDCA). Results are shown as nmol/g cell protein; means _+ SD
(n = 4). the protective effect of UDCA at these concentrations, which are above the critical micellar bile acid concentration, might be explained by an effect on micelle composition: UDCA could favor the formation of simple micelles and isolated bile acid monomers, which have a lesser detergent action on cholesterol-rich cell membranes than that of mixed micelles. 23 Also studying cultured h u m a n hepatocytes, Galle et a124 found that UDCA reduced the toxic effect of glyco CDCA suggesting a direct cytoprotective effect. The disagreement with our data could stem from differences in methodology or in the expression of the results. In particular, we expressed LDH leakage as a percentage of total (intracellular and extracellular) LDH activity and not as the quantity present in supernatant. We should not exclude that our failure to detect a cytoprotective effect of UDCA was caused by cell dedifferentia-
FIG. 7. Effect of CDCA alone and in the presence of TUDCA on albumin secretion by h u m a n hepatocytes in primary culture after 24 hours' incubation. TUDCA failed to reverse the decrease in albumin synthesis induced by 100 ~mol/L CDCA. Values are means _+ SD (n = 5). *P < .05 vs. controls. **P < .05 vs. controls, but not vs. CDCAtreated cells.
tion; however, like other authors, 25 we used h u m a n hepatocytes 48 hours after their isolation, i.e., the time required for the formation of cell-cell contacts necessary for exchanges and the expression of differentiation markers. By way of an example, we have found that albumin secretion increases by 30% between days 1 and 3 of culture. Moreover, in our culture conditions, bile acids are conjugated by hepatocytes (Fig. 1). Both functions are hepatospecific. In agreement with our findings other teams have reported a lack of cytoprotection by UDCA. 19'26 Our data tend to support the notion that the therapeutic effect of UDCA in chronic cholestatic diseases is related to qualitative and quantitative changes in the endogenous circulating bile acid pool as a result of reduced active ileal absorption, although this latter mechanism does not take into account UDCA-induced protection against the cholestasis and necrosis caused by hydrophobic bile acids in perfused rat liver. 5 The w a y in which UDCA protects the liver in cholestatic liver disease is unknown. Theoretically, the hepatoprotective effect of UDCA can be explained by a decrease in the concentration of endogenous bile acids bathing liver cells, or by a direct effect of UDCA itself (or one of its conjugated forms) on hepatocyte or nonhepatocytic cell viability and function. UDCA could thus also have a direct protective effect by promoting canalicular efflux of other bile acids. This mechanism is supported by the protective effect of UDCA in the rat liver loaded with hydrophobic bile acids, s a situation in which UDCA increases bile acid elimination. It has been generally assumed that this finding was related to the cytoprotective effect of UDCA on hepatocytes. However, one alternative is that UDCA, a bile acid with very high intrinsic hepatic clearance 27 increases the vectorial transport of bile acids by facilitating intracellular or canalicular transport. Recently, Ha~ssinger et al 2s have provided circumstantial evidence that increased
HEPATOLOGYVol. 22, No. 1, 1995
taurocholate intrinsic clearance induced by UDCA is mediated by hepatocellular swelling. Although canalicular elimination of bile acids is theoretically possible in primary hepatocyte cultures, 29 it would occur in a closed system, and neither intracellular nor extracellular concentrations would be affected; this could explain the lack of protection by UDCA in this model. Despite these results, a hepatoprotective effect of UDCA cannot be ruled out. Indeed, UDCA could preserve other liver cells or improve hepatocyte functions other than those studied here. In conclusion, CDCA shows concentration-dependent toxicity for cultured human hepatocytes, an effect that is not reversed by UDCA or TUDCA. The presence of polarized hepatocytes or an intact enterohepatic circulation seems to be required for the hepatoprotective effect of UDCA to become apparent. REFERENCES
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