- Email: [email protected]
Retrograde injections of formaldehyde into the biliary tree induce alterations of biliary epithelial function in rats
Retrograde Injections of Formaldehyde Into the Biliary Tree Induce Alterations of Biliary Epithelial Function in Rats MICHELINE DUMONT,1 CORINNE D’HONT,1 ALAIN MOREAU,2 HERMINE MBAPE,2 GE´RARD FELDMANN,2
Formaldehyde may induce severe lesions of intrahepatic and extrahepatic bile ducts. The purpose of this study was to examine in vivo the functional consequences of an alteration of the biliary epithelium induced by a retrograde intrabiliary injection of formaldehyde in rats. After basal bile collection, a 10% formaldehyde solution was injected into the biliary tree of anesthetized rats, and the cannula was occluded for 30 minutes. Choleresis was then reestablished, and bile flow, bile acid, and bicarbonate secretion were measured both spontaneously and during ursodeoxycholate infusions. Formaldehyde injections induced a significant increase in bile flow and a marked inhibition of ursodeoxycholate-induced increase in biliary bicarbonate concentration and secretion. Biliary glucose secretion, which is normally very low, was increased about 20fold in animals injected with formaldehyde. Histological and ultrastructural examination of the liver showed alterations of biliary epithelial cells, whereas hepatocytes, bile canaliculi, and canalicular tight junctions remained normal. Hepatocytic excretory function, as assessed by biliary secretion of bile acids, was not affected. It was concluded that short-term formaldehyde intrabiliary injections cause an inhibition of ursodeoxycholate-induced hypersecretion of bicarbonate, an increase in biliary glucose secretion, and selective structural alterations of biliary epithelial cells. These results suggest that formaldehyde retrograde biliary injection may be a useful model to study alterations of biliary epithelial function in vivo. (HEPATOLOGY 1996;24:1217-1223.) Bile formed by the hepatocytes (canalicular bile) is modified by the bile ductules and ducts on its way to the duodenum.1 Both absorptive and secretory processes have been described previously.1 The net effect is the addition of a bicarbonate-rich solution.2 Recently, the cellular and molecular mechanisms of secretion have been characterized: an adenosine 3*,5*-cyclic monophosphate–stimulated chloride channel, the cystic fibrosis transmembrane conductance regulator protein, has been identified on the apical membrane of rat and human biliary epithelial cells.3 The channel allows the extrusion of chloride ions that probably stimulate bicarbonate secretion through activation of a chloride/bicarbonate exchanger.4
Abbreviations: UDCA, ursodeoxycholic acid; AST, aspartate transaminase; LDH, lactate dehydrogenase. From the 1Unite´ de Recherches de Physiopathologie He´patique (INSERM Unite´ 24 and Association Claude Bernard) and Service d’He´patologie, Hoˆpital Beaujon, Clichy, France; and 2Unite´ de Recherches Structure et Fonctions des Cellules He´patiques (INSERM Unite´ 327), Faculte´ de Me´decine Xavier Bichat, Paris, France. Received December 8, 1994; accepted June 27, 1996. Address reprint requests to: Serge Erlinger, M.D., INSERM Unite´ 24, Hoˆpital Beaujon, 100 Bd du Ge´ne´ral Leclerc F-92118 Clichy Cedex, France. Copyright q 1996 by the American Association for the Study of Liver Diseases. 0270-9139/96/2405-0039$3.00/0
Hepa 0034
/
5P16$$$661
10-09-96 15:03:25
SERGE ERLINGER1
Biliary channels may also play a role in ursodeoxycholateinduced hypercholeresis.5-7 The most widely accepted hypothesis to account for bicarbonate-rich hypercholeresis induced by ursodeoxycholate is the so-called cholehepatic cycle,8 which implies passive absorption of protonated ursodeoxycholic acid (UDCA) through the biliary epithelial cells. Finally, biliary ducts play a role in the reabsorption of a number of organic compounds, including amino acids9 and glucose.10 Alteration of these functions are likely to occur in diseases affecting the biliary epithelium, such as primary biliary cirrhosis or primary sclerosing cholangitis. In this study, our purpose was to examine in vivo the functional consequences of an alteration of the biliary epithelium by a retrograde intrabiliary injection of formaldehyde. This agent has been shown to induce a secondary cholangitis in rats,11 dogs,12 and humans.13 We examined the immediate consequences of intrabiliary injections of formaldehyde on ursodeoxycholate-induced hypercholeresis and on biliary glucose secretion. We showed that ursodeoxycholate-induced hypercholeresis was decreased but not suppressed and that glucose biliary secretion was considerably increased. Hepatocyte excretory function, as assessed by biliary secretion of bile acids, was not affected. Thus, retrograde biliary injection of formaldehyde may be a useful model to study biliary function in vivo. MATERIALS AND METHODS Chemicals. UDCA, tauro-UDCA, and ethylcarbamate were pur-
chased from Sigma Chemical Co. (St. Louis, MO). Pentobarbital was purchased from Sanofi (Paris, France), and formaldehyde, 1-propanol, propionic acid, and isoamyl acetate were purchased from Merck (Darmstadt, Germany). Experimental Procedures. Male Sprague-Dawley rats (CD strain; Charles River Breeding Laboratories, St. Aubin-les-Elbeuf, France), weighing 243 { 20 g (mean { SD), were used. They were anesthetized intraperitoneally with pentobarbital (5 mg/100 g body wt). Rectal temperature was monitored and maintained at 387C { 0.57C on a heating table. Tracheostomy was performed. A jugular vein was cannulated with a polyethylene catheter no. 3 (ID, 0.58 mm; Biotrol, Paris, France), and the common bile duct was cannulated with a no. 1 catheter (ID, 0.3 mm; Biotrol). Bile was collected immediately after cannulation during three periods of 10 minutes. The mean of the two last periods was taken as basal values for bile flow. Then, a retrograde intrabiliary injection14 of 10% formaldehyde was performed in a volume of 40 mL/100 g body wt, corresponding to the volume of the biliary tree,15 and 10 mL for the cannula dead space. The bile duct cannula was occluded for 30 minutes. Control rats were administered 0.15 mol/L NaCl instead of formaldehyde. After 30 minutes, the cannula was opened, and bile flow was collected during two periods of 15 minutes to obtain a stable bile flow. Then bile was collected every 10 minutes for 90 minutes. In two other groups, rats were treated as described previously with 10% formaldehyde or NaCl and were administered an infusion of UDCA or tauro-UDCA at the rate of 800 nmolrmin01r100 g body wt01 for 90 minutes after the 30 minutes of bile flow stabilization. The mean of the two last periods was taken as UDCA values. Two additional groups of rats were studied to measure the bile/ plasma concentration ratio of sucrose. After ligation of renal pedicles, 5 mCi of [3H]sucrose (Amersham Radiochemicals, Amersham, England) was injected into the penile vein. After a period of equilibration of 60 minutes, the carotid artery and the common bile duct were
1217
AID
AND
hpta
WBS: Hepatology
1218
DUMONT ET AL.
HEPATOLOGY November 1996
FIG. 1. Experimental design.
cannulated. Bile samples were collected during 10-minute periods for 30 minutes. Thereafter, a retrograde intrabiliary injection of 10% formaldehyde (n Å 6) or NaCl (n Å 6) was performed, and the catheter was occluded for 30 minutes. Then the cannula was opened, and bile was collected for 30 minutes. Aliquots of 200 mL of arterial blood were taken at the midpoint of bile collection periods before and 60 minutes after the reopening of the bile duct cannula. The experimental design is shown in Fig. 1. Four adult Sprague-Dawley rats were prepared specifically for morphological investigation. Two rats were administered a retrograde intrabiliary injection of 10% formaldehyde, whereas two other rats were administered 0.15 mol/L NaCl instead of formaldehyde and were used as controls. The experimental procedure for these four rats was identical to that followed for the other rats: the bile duct cannula was occluded for 30 minutes and then reopened. The four rats were killed 30 minutes after the reopening of the cannula. Analytical Methods. Bile acid concentration was measured by the enzymatic method using 3a-hydroxysteroid dehydrogenase.16 Biliary concentration of glucose was measured with a glucose oxidase method using a kit phenol-amino-4-antipyrine-peroxidase (bioMerieux, Marcy l’Etoile, France). Biliary concentration of HCO0 3 was measured with a carbon dioxide analyzer (Corning 965; Ciba Corning Diagnostics, Halstead, England). Biliary bile acids (tauroursodeoxycholate, glycoursodeoxycholate, and ursodeoxycholate) were separated by thin-layer chromatography.17 Bile was deposited on 0.25mm silica gel 60 F254 plates (Merck). The solvent system used was as follows: water/1-propanol/propionic acid/isoamyl acetate (3:4:6:6, vol/vol). The plates were sprayed with water, and the spots were eluted in tetrasodium diphosphate solution (0.1 mol/L). Bile acid concentration was measured in each eluate with 3a-hydroxysteroid dehydrogenase.18 Standards of ursodeoxycholate, tauroursodeoxycholate, and glycoursodeoxycholate were used. For measurement of 3H activity, aliquots of 50 mL of plasma or bile were put into a glass vial, and 10 mL of scintillation medium (ACS; Amersham Radiochemicals) was added. The activity was measured in a liquid scintillation spectrometer (Minaxi, Tricarb 4000 series; Packard, Meriden, CT) with automatic correction for quenching. Bile/plasma ratio of [3H]sucrose activity was calculated. Serum aspartate transaminase (AST) and lactate dehydrogenase (LDH) activities were determined by standard enzymatic methods (Sigma Diagnostic and Biotrol kits, respectively). Histological and Ultrastructural Examination. For each rat, a part of the common bile duct and two liver fragments were immediately taken after the animal was killed. Liver fragments were obtained in the hilar region and at the periphery of the organ. Bile duct and liver fragments were divided in two parts for histological and electron microscopic examination. The part designed for histological examination was fixed in Bouin’s fluid and embedded in paraffin. Five-micrometer-thick sections were stained with hematoxylin-eosin and examined on light microscopy. The part designed for electron microscopic examination was cut immediately into 1-mm-thick blocks, and the blocks were fixed in a 2.5% solution of glutaraldehyde buffered with 0.1 mol/L phosphate buffer, pH 7.4, for 2 hours at 47C. After washing in phosphate buffer, the blocks were postfixed in a 1% solution of osmium tetroxide buffered with veronal buffer, pH 7.2, at room temperature for 1 hour. Blocks were dehydrated by graded alcoholic solutions and embedded in epoxy resins. Semithin sections stained with toluidine blue were made on each block to localize the bile ducts. At least three bile duct–containing blocks were cut for the ultrastructural examination of the common bile duct
AID
Hepa 0034
/
5P16$$$662
10-09-96 15:03:25
FIG. 2. Effect of formaldehyde on spontaneous and ursodeoxycholate-induced bile flow in the rat. UDCA or tauro-UDCA were infused at a rate of 800 nmolrmin01r100 g01. Each point represents the mean { 1SD. ●, Formaldehyde plus UDCA; j, formaldehyde plus tauro-UDCA; m, formaldehyde; s, NaCl plus UDCA; and n, NaCl.
and of each liver fragment. Ultrathin sections stained with uranyl acetate and lead citrate were analyzed with a JEOL 1010 electron microscope. All animals received humane care in compliance with international recommendations. Statistical Analysis. Results are expressed as means { SD. Differences between mean values were assessed statistically using the Student’s t test. RESULTS Effect of Formaldehyde on Spontaneous and Ursodeoxycholate-Induced Choleresis. After retrograde injection of formal-
dehyde (n Å 7), spontaneous bile flow was significantly greater (11.1 { 1.1 mLrmin01r100 g01) than in rats with a retrograde injection of NaCl (n Å 6) (7.3 { 1.6 mLrmin01r100 g01; P õ .001). In contrast, during infusion of ursodeoxycholate, bile flow was similar in rats administered formaldehyde (n Å 6) and in rats administered NaCl (n Å 6) (19.7 { 4.0 and 20.4 { 4.0 mLrmin01r100 g01; NS) (Fig. 2). In rats administered formaldehyde, bile flow during infusion of tauroursodeoxycholate was significantly less (13.9 { 2.4 mLrmin01r100
FIG. 3. Effect of formaldehyde on spontaneous and ursodeoxycholate-induced bile acid output in the rat. UDCA or tauro-UDCA were infused at a rate of 800 nmolrmin01r100 g01. Each point represents the mean { 1SD. ●, Formaldehyde plus UDCA; j, formaldehyde plus tauro-UDCA; m, formaldehyde; s, NaCl plus UDCA; and n, NaCl.
hpta
WBS: Hepatology
HEPATOLOGY Vol. 24, No. 5, 1996
DUMONT ET AL.
FIG. 4. Effect of formaldehyde on spontaneous and ursodeoxycholate-induced bicarbonate output in the rat. UDCA or tauro-UDCA were infused at a rate of 800 nmolrmin01r100 g01. Each point represents the mean { 1SD. ●, Formaldehyde plus UDCA; j, formaldehyde plus tauro-UDCA; m, formaldehyde; s, NaCl plus UDCA; and n, NaCl.
g01) than that observed during ursodeoxycholate infusion (19.7 { 4.0 mLrmin01r100 g01; P õ .05). Bile salt output was similar in animals injected with NaCl or formaldehyde and infused with ursodeoxycholate or tauroursodeoxycholate (Fig. 3). Because it is known that hypercholeresis induced by ursodeoxycholate is associated with an increase in bicarbonate concentration and secretion into bile,5 we measured biliary bicarbonate concentration and output in rats injected with NaCl and rats administered formaldehyde. As expected, in rats administered NaCl, bicarbonate concentration and output increased significantly during ursodeoxycholate infusion (concentration, 47.3 { 9.2 against 30.8 { 3.9 mmolrL01; P õ .01; output, 990.5 { 377.6 against 227.8 { 64.4 nmolr min01r100 g01; P õ .001; increase in bicarbonate output, 762 nmolrmin01r100 g01 or 334%). Bicarbonate concentration and output also increased in animals injected with formaldehyde (concentration, 35.1 { 4.1 against 28.5 { 1.6 mmolrL01; P õ .01; output, 694.7 { 188.1 against 316.4 { 47.2 nmolr min01r100 g01; P õ .001). However, the increase in bicarbon-
1219
FIG. 6. Effect of formaldehyde (j) on biliary excretion of ursodeoxycholate and its conjugates in the rat. UDCA was infused at a rate of 800 nmolr min01r100 g01. The concentrations of UDCA, tauro-UDCA, and glyco-UDCA in bile (expressed in percentage of total bile acids) were measured by thinlayer chromatography at 40, 60, and 90 minutes after the beginning of UDCA infusion. Each column represents the mean { 1SD. h, NaCl.
ate output was significantly less (379 nmolrmin01r100 g01 or 120%) than that of animals injected with NaCl (P õ .001) (Fig. 4). Bicarbonate concentration and output were not modified by tauroursodeoxycholate (Fig. 4). Effect of Formaldehyde on Biliary Glucose Secretion. Biliary glucose concentration (10.1 { 3.2 against 0.9 { 0.1 mmolrL01; P õ .001) and output (110.8 { 31.5 against 6.3 { 5.0 nmolrmin01r100 g01; P õ .001) were significantly greater in rats injected with formaldehyde than in those injected with NaCl. Similarly, during infusion of ursodeoxycholate, glucose biliary concentration (8.1 { 1.9 against 1.3 { 0.5 mmolrL01; P õ .001) and output (160.1 { 55.7 against 26.2 { 8.1 nmolr min01r100 g01; P õ .001) were significantly greater in rats injected with formaldehyde than in those injected with NaCl (Fig. 5). Effect of Formaldehyde on Biliary Excretion of Ursodeoxycholate and its Conjugates. To test whether the inhibition of
hypercholeresis and biliary bicarbonate secretion in formaldehyde-injected rats was caused by an alteration of the putative cholehepatic cycling,8 we measured the biliary excretion of unconjugated ursodeoxycholate. The percentage of unconjugated ursodeoxycholate and that of glyco- and tauro-conjugated ursodeoxycholate were similar in formaldehyde-injected animals and NaCl-injected controls (Fig. 6). Effect of Formaldehyde on [3H]Sucrose Bile/Plasma Ratio.
Before retrograde injection, basal bile/plasma ratios of [3H]sucrose were similar in the two groups (0.20 { 0.05 and 0.16 { 0.04). After retrograde intrabiliary injection, the bile/ plasma ratio was significantly greater in formaldehyde-injected rats (0.51 { 0.09) than in NaCl-injected rats (0.22 { 0.03; P õ .001). Effect of Formaldehyde on Serum Enzyme Activities. There was no difference in basal values of AST and LDH activities
TABLE 1. Serum Activities of AST and LDH Before and After Retrograde Injection of Formaldehyde or NaCl Into the Biliary Tree Retrograde Biliary Injection NaCl (n Å 6)
FIG. 5. Effect of formaldehyde on biliary glucose secretion in the rat. UDCA or tauro-UDCA were infused at a rate of 800 nmolrmin01r100 g01. Each point represents the mean { 1SD. ●, Formaldehyde plus UDCA; j, formaldehyde plus tauro-UDCA; m, formaldehyde; s, NaCl plus UDCA; and n, NaCl.
AID
Hepa 0034
/
5P16$$$662
10-09-96 15:03:25
Formaldehyde (n Å 6)
Enzyme Activities
Before
After
Before
After
AST (U/L) LDH (U/L)
76 { 11 356 { 83
171 { 45 581 { 131
80 { 27 309 { 81
131 { 61 436 { 159
hpta
WBS: Hepatology
1220
DUMONT ET AL.
AID
Hepa 0034
HEPATOLOGY November 1996
/
5P16$$0034
10-09-96 15:03:25
hpta
WBS: Hepatology
HEPATOLOGY Vol. 24, No. 5, 1996
DUMONT ET AL.
1221
FIG. 7. Histological and ultrastructural appearance of biliary cells in (A, C, and E) control rats and (B, D, and F) formaldehyde-treated rats. In control rats, note the normal structure of (A) the biliary cells of the common bile duct and of (C and E) a bile duct localized in a hilar portal space of the liver. The biliary epithelium [e] is composed of (A) cylindric or (C and E) cubic cells regularly arranged around the lumen [L] of the bile duct. (E) At the ultrastructural level, few organelles are visible in the cytoplasm of the cells. (B, D, and F) In formaldehyde-treated rats, the biliary epithelium [e] of the common bile duct is (B) separated from the surrounding connective tissue in some places. (B and D) Biliary cells are shrunken, and their nuclei and cytoplasm are difficult to recognize. At the ultrastructural level, note the (F) irregular shape of biliary cell nuclei, the absence of microvilli in front of the lumen [L], and the electrondense appearance of the cytoplasm (original magnification: A and B, 1501; C and D, 9001; E, 6,0001; and F, 7,0001). b
between the two groups. After retrograde injection of formaldehyde or NaCl, the serum activities of AST and LDH were greater than the basal activities. However, there was no significant difference between formaldehyde- and NaCl-injected animals (Table 1). Light Microscopy. In the control rats administered saline, the histological appearance of the biliary epithelial cells was normal either in the common bile duct (Fig. 7A) or in the portal bile ducts (Fig. 7C) localized in the hepatic hilar region. No alteration of these cells was observed in the bile ducts of the portal spaces visible in the peripheral regions of the liver. No inflammatory cells were present around the biliary ducts, whatever their localization. Hepatocytes were normal in the two regions of the liver examined. In the rats administered formaldehyde, the biliary epithelium was deeply altered. In the common bile duct, it was separated from the surrounding connective tissue by more or less large spaces (Fig. 7B). Moreover, the biliary cells were so shrunken that it was difficult to recognize their nucleus and cytoplasm. Similar changes were observed in the biliary ducts of the portal spaces localized either in the hepatic hilar regions (Fig. 7D) or at the periphery of the liver. However, alterations of bile duct cells were not visible in all of the examined portal spaces. In particular, some bile ducts of the peripheral portal spaces were normal or almost normal. Whatever the localization of the bile ducts, no inflammatory cells were observed around the biliary cells (Figs. 7B and D). In the liver, hepatocytes were normal around the portal spaces (Fig. 7D). Electron Microscopy. No alteration of the biliary cells was observed in control rats administered saline. A typical aspect of a bile duct from a hilar portal space is shown in Fig. 7E. Biliary cells with a prominent nucleus and a scanty cytoplasm were regularly arranged along the lumen of the bile ducts. At a greater magnification (Fig. 8A), intact tight junctions were visible. Microvilli were visible at the apical plasma membrane domain of the cells. A continuous basement membrane surrounded the biliary cells. No inflammatory cells were present in the connective tissue. The ultrastructural appearance of periportal hepatocytes was normal, and intact tight junctions (Fig. 8C) were observed at the level of bile canaliculi. In rats administered formaldehyde, the ultrastructural appearance of biliary cells was very often different from that observed in control rats. A typical aspect of a bile duct from a hilar portal space is shown in Fig. 7F. The nucleus of the biliary cells was very altered with an irregular size and profound invaginations. The cytoplasm was electron dense and shrunken. Cytoplasmic organelles were difficult to recognize. Microvilli were absent at the apical plasma membrane domain. At a greater magnification (Fig. 8B), tight junctions were unaltered. Similar ultrastructural aspects were observed in the biliary cells from the other parts of the bile ducts. However, some bile ducts were normal, in particular those localized in peripheral portal spaces. Finally, the ultrastructural appearance of periportal hepatocytes was normal, and intact tight junctions were observed around bile canaliculi (Fig. 8D). As in control rats, no inflammatory cells were visible around the bile ducts, whatever their localization in the biliary tract or in the liver.
AID
Hepa 0034
/
5P16$$$662
10-09-96 15:03:25
DISCUSSION
In these experiments, we attempted to impair selectively the function of the biliary epithelium by a retrograde intrabiliary injection of formaldehyde in rats in vivo. The first observation in this study was a significant inhibition of ursodeoxycholate-induced hypersecretion of bicarbonate in the rat. In this animal, ursodeoxycholate, when administered in the unconjugated form, induces a greater bile flow than tauroursodeoxycholate or taurocholate.5,6 This hypercholeresis is associated with an increase in biliary bicarbonate concentration and secretion.5,6 This phenomenon has been accounted for by the secretion of ursodeoxycholate in the unconjugated form in canalicular bile followed by its protonation (with generation of bicarbonate) and passive reabsorption by the biliary epithelial cells8 and its return to the liver by the peribiliary plexuses. At each of these cholehepatic cycles, a bicarbonate ion is generated, explaining the increase in biliary bicarbonate concentration and secretion. In rats injected with formaldehyde, we observed a significant inhibition of ursodeoxycholate-induced bicarbonate output (Fig. 4). However, passive absorption of ursodeoxycholate was not affected because the proportion of unconjugated ursodeoxycholate in the final bile was unchanged (Fig. 6). If cholehepatic cycling had been interrupted, we would have expected an increase in unconjugated ursodeoxycholate in the bile. The decrease in bicarbonate generation (and, consequently, of bicarbonate-induced bile flow) may be explained by an inhibition of luminal carbonic anhydrase by formaldehyde. The protonation of ursodeoxycholate is caused by subtraction of H/ from carbonic acid, thereby producing an increase in biliary HCO0 3 concentration. Because the CO2 -H2CO3 buffer is an open system and CO2 freely crosses biological membranes, H2CO3 is formed by luminal carbonic anhydrase.19 Inhibition of this enzyme may lead to decreased H2CO3 generation and availability and, thus, to decreased HCO0 3 formation. Two pieces of evidence support this interpretation. First, previous studies from this and other laboratories have shown that acetazolamide, a known inhibitor of carbonic anhydrase, also inhibited the stimulation of bicarbonate secretion induced both by ursodeoxycholate and secretin.20,21 Second, glutaraldehyde, which has chemical similarities with formaldehyde, is known to inhibit carbonic anhydrase.22 Studies of isolated biliary epithelial cells may help to test this hypothesis. The second observation in this study is a marked increase in glucose biliary secretion in formaldehyde-injected rats. In the normal state, biliary glucose concentration is remarkably low. This has been explained by a passive glucose entry into canalicular bile followed by an active glucose reabsorption by the biliary epithelial cells.23 This reabsorption seems to be mediated by two luminal glucose transport systems: a sodium-dependent and a sodium-independent system.23 Our results are consistent with an inhibition of these transport systems by formaldehyde, resulting in increased glucose output. In turn, this increased glucose output should result in an increased bile flow by an osmotic mechanism. Our observations are again consistent with this hypothesis. In rats injected with formaldehyde, basal bile flow was increased significantly. Glucose output was also increased. We propose that the most likely explanation for this increase in bile flow is an osmotic effect of glucose. Similarly, when UDCA was
hpta
WBS: Hepatology
1222
DUMONT ET AL.
HEPATOLOGY November 1996
FIG. 8. Ultrastructural appearance of tight junctions [TJ] of biliary cells and hepatocytes in (A and C) control rats and (B and D) formaldehyde-treated rats. The junctions appear morphologically normal in the two groups of rats, not only between the biliary cells (observed here in a bile duct from a hepatic hilar portal space) but also in periportal hepatocytes (A, 28,0001; B, 16,0001; C, 34,5001; and D, 32,0001). N, nucleus; H, hepatocyte; BC, bile canaliculus; mv, microvilli.
infused in rats injected with formaldehyde, ursodeoxycholate-induced HCO0 3 secretion was significantly less than that in NaCl-injected rats, but bile flow was virtually identical. The most likely explanation is that glucose was the osmotically active compound responsible for the increase in basal bile flow and the maintenance of bile flow under ursodeoxycholate infusion. Assuming an osmotic activity of 3-10 mL/ mmol23 and taking into account a biliary secretion of glucose of 0.11 mmolrmin01r100 g01, the glucose-induced bile flow should be 0.33-1.1 mLrmin01r100 g01. The observed increase in bile flow (11 mLrmin01r100 g01 in formaldehyde-injected rats vs. 7.3 mLrmin01r100 g01 in NaCl-injected controls) is far greater than the calculated value. This was also the case in the experiments by Lira et al.,23 where the amount of fluid reabsorbed in the bile ducts exceeded that predicted from the amount of glucose reabsorbed. We have to postulate, as did Lira et al., that reabsorption of other substrates (such as amino acids or inorganic electrolytes) is also inhibited by
AID
Hepa 0034
/
5P16$$$663
10-09-96 15:03:25
formaldehyde. A possible substrate could be glutathione: glutathione is secreted into bile by the hepatocytes and cleaved in the biliary lumen into several peptides by g-glutamyl transferase.24 These peptides are reabsorbed in the biliary tree. g-Glutamyl transferase is inhibited by incubation with formaldehyde (data not shown); this would result in an increased biliary secretion of glutathione. An alternative explanation for the increase in biliary glucose concentration and secretion may be that formaldehyde increased the permeability of the biliary epithelium to glucose. Because glucose concentration in plasma is greater than in bile, an increase in biliary permeability could facilitate the movement of glucose from plasma to bile. To test this possibility, we estimated the permeability between blood and bile by measuring the bile/plasma concentration ratio of sucrose.25 We found a significant increase in the bile/plasma ratio after formaldehyde injection: the bile/plasma ratio was greater in rats administered formaldehyde than in the NaCl-
hpta
WBS: Hepatology
HEPATOLOGY Vol. 24, No. 5, 1996
DUMONT ET AL.
injected controls. This indicates that the permeability to sucrose was increased. Therefore, it is likely that the permeability to glucose was also increased. Theoretically, this increase in permeability may be caused by an alteration: of the hepatocytes; of the tight junctions around bile canaliculi; of the biliary epithelial cells; or of tight junctions between biliary cells. Therefore, we performed a histological and ultrastructural study. The results indicated that the hepatocytes and tight junctions around bile canaliculi were always normal. There were alterations of the intrahepatic biliary epithelial cells, particularly of their apical membrane. However, tight junctions between these cells appeared to be morphologically intact. The most striking alterations were located on the extrahepatic bile ducts. The extrahepatic bile ducts appear to be the most likely site of the increased permeability. Finally, we present evidence that the hepatocytes were functionally and morphologically intact after formaldehyde injection. First, bile acid secretion after ursodeoxycholate and tauroursodeoxycholate infusions was normal. This indicates a normal hepatocyte excretory function. Secondly, hepatocyte enzymes (AST and LDH activities) were only moderately elevated after intrabiliary injection and not significantly different between formaldehyde- and NaCl-injected animals. Thirdly, the ultrastructural appearance of hepatocytes and hepatocyte tight junctions was always normal. Toxicity of formaldehyde on the liver has been reported in the literature,26,27 mostly on the isolated perfused rat liver and isolated rat hepatocytes. This was apparently not the case in our experimental system, perhaps because formaldehyde was injected in the biliary tree rather than in the circulation or directly in contact with hepatocytes. None of the ultrastructural lesions previously reported (mitochondrial damage or alterations of the rough endoplasmic reticulum) was observed in this study. In summary, formaldehyde intrabiliary injection resulted in an inhibition of ursodeoxycholate-induced bicarbonate secretion in rats and in a marked increase in biliary glucose. These observations are consistent with a profound and selective alteration of biliary epithelial function by formaldehyde. The alteration was selective because the excretory function and morphology of hepatocytes were normal. Alteration of biliary epithelial function was probably caused by both inhibition of transport systems of biliary epithelial cells and destruction of cells of the extrahepatic bile ducts, resulting in increased biliary permeability. Intrabiliary injection of formaldehyde may be a useful model to study alterations of biliary epithelial function in vivo.
4. 5. 6. 7.
8.
9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24.
REFERENCES 1. Erlinger S. Secretion of bile. In: Schiff E, Schiff L, eds. Diseases of the liver. Philadelphia: Lippincott, 1993:85-107. 2. Tarsetti F, Lenzen R, Salvi R, Schuler E, Dembitzer R, Tavoloni N. Biology and pathobiology of intrahepatic biliary epithelium. In: Tavoloni N, Berk PD, eds. Hepatic transport and bile secretion: physiology and pathophysiology. New York: Raven, 1993:619-635. 3. Cohn JA, Strong TV, Picciotto MR, Nairn AC, Collins FS, Fitz JG. Localiza-
AID
Hepa 0034
/
5P16$$$663
10-09-96 15:03:25
25. 26. 27.
1223
tion of the cystic fibrosis transmembrane conductance regulator in human bile duct epithelial cells. Gastroenterology 1993;105:1857-1864. Fitz JG, Basavappa S, McGill J, Melhus O, Cohn JA. Regulation of membrane chloride current in rat bile duct epithelial cells. J Clin Invest 1993; 91:319-328. Dumont M, Erlinger S, Uchman S. Hypercholeresis induced by ursodeoxycholic acid and 7-ketolithocholic acid in the rat: possible role of bicarbonate transport. Gastroenterology 1980;79:82-89. Kitani K, Kanai S. Effect of ursodeoxycholate on the bile flow in the rat. Life Sci 1982;31:1973-1985. Gurantz D, Schteingart CD, Hagey LR, Steinbach JH, Grotmol T, Hofmann AF. Hypercholeresis induced by unconjugated bile acid infusion correlates with recovery in bile of unconjugated bile acids. HEPATOLOGY 1991;13:540-550. Yoon YB, Hagey LR, Hofmann AF, Gurantz D, Michelotti EL, Steinbech JH. Effect of side-chain shortening on the physiological properties of bile acids: hepatic transport and effect on biliary secretion of 23-nor-ursodeoxycholate in rodents. Gastroenterology 1986;90:837-852. Ballatori N, Jacob R, Barrett C, Boyer JL. Biliary catabolism of glutathione and differential reabsorption of its amino acid constituents. Am J Physiol 1988;254:G1-G7. Guzelian P, Boyer J. Glucose reabsorption from bile, evidence for biliohepatic circulation. J Clin Invest 1974;53:526-535. Houry S, Languille O, Huguier M, Benhamou JP, Belghiti J, Msika S. Sclerosing cholangitis induced by formaldehyde solution injected into the biliary tree of rats. Arch Surg 1990;125:1059-1061. Grimaud JC, Sastre B, Orsoni P, Monges G, Treffot MJ, Monges A, Michotey G. Le roˆle du formol dans la survenue de le´sions scle´rosantes des voies biliaires. J Chir (Paris) 1984;121:163-169. Belghiti J, Benhamou JP, Houry S, Grenier P, Huguier M, Fe´ke´te´ F. Caustic sclerosing cholangitis. A complication of the surgical treatment of hydatid disease of the liver. Arch Surg 1986;121:1162-1165. Farges O, Corbic M, Dumont M, Maurice M, Erlinger S. Permeability of the rat biliary tree to ursodeoxycholic acid. Am J Physiol 1989;256:G653G660. Barber-Riley G. Measurement of capacity of biliary tree in rats. Am J Physiol 1963;205:1122-1126. Berthelot P, Erlinger S, Dhumeaux D, Preaux AM. Mechanism of phenobarbital-induced hypercholeresis in the rat. Am J Physiol 1970;219:809813. Hofmann AF. Thin-layer adsorption chromatography of free and conjugated bile acids on silicic acid. J Lipid Res 1962;3:127-128. Turnberg LA, Anthony-Mote A. The quantitative determination of bile salts in bile using thin-layer chromatography and 3a-hydroxysteroid dehydrogenase. Clin Chim Acta 1969;24:253-259. Strazzabosco M, Sakisaka S, Hayakawa T, Boyer J. Effect of UDCA on intracellular and biliary pH in isolated rat hepatocyte couplets and perfused livers. Am J Physiol 1991;260:G58-G69. Garcia-Marin JJ, Dumont M, Corbic M, de Couet G, Erlinger S. Effect of acid-base balance and acetazolamide on ursodeoxycholate-induced biliary bicarbonate secretion. Am J Physiol 1985;248:G20-G27. Buanes T, Grotmol T, Landsverk T, Ridderstra˚le Y, Ræder MG. Importance of carbonic anhydrase for canalicular and ductular choleresis in the pig. Acta Physiol Scand 1988;133:535-544. Buanes T, Grotmol T, Landsverk T, Ridderstra˚le Y, Ræder MG. Histochemical localization of carbonic anhydrase in the pig’s exocrine pancreas. Acta Physiol Scand 1986;128:437-444. Lira M, Schteingart CD, Steinbach JH, Lambert K, McRoberts JA, Hofmann AF. Sugar absorption by the biliary ductular epithelium of the rat: evidence for two transport systems. Gastroenterology 1992;102:563-571. Ballatori N, Jacob R, Barrett C, Boyer JL. Biliary catabolism of glutathione and differential reabsorption of its amino acid constituents. Am J Physiol 1988;254:G1-G7. Forker EL. The effect of estrogen on bile formation in the rat. J Clin Invest 1969;48:654-663. Ku RH, Billings RE. Relationships between formaldehyde metabolism and toxicity and gluthatione concentrations in isolated rat hepatocytes. Chem Biol Interact 1984;51:25-36. Strubelt O, Younes M, Pentz R. Mechanistic study on formaldehyde-induced hepatotoxicity. J Toxicol Environ Health 1989;27:351-366.
hpta
WBS: Hepatology
Formaldehyde may induce severe lesions of intrahepatic and extrahepatic bile ducts. The purpose of this study was to examine in vivo the functional consequences of an alteration of the biliary epithelium induced by a retrograde intrabiliary injection of formaldehyde in rats. After basal bile collection, a 10% formaldehyde solution was injected into the biliary tree of anesthetized rats, and the cannula was occluded for 30 minutes. Choleresis was then reestablished, and bile flow, bile acid, and bicarbonate secretion were measured both spontaneously and during ursodeoxycholate infusions. Formaldehyde injections induced a significant increase in bile flow and a marked inhibition of ursodeoxycholate-induced increase in biliary bicarbonate concentration and secretion. Biliary glucose secretion, which is normally very low, was increased about 20fold in animals injected with formaldehyde. Histological and ultrastructural examination of the liver showed alterations of biliary epithelial cells, whereas hepatocytes, bile canaliculi, and canalicular tight junctions remained normal. Hepatocytic excretory function, as assessed by biliary secretion of bile acids, was not affected. It was concluded that short-term formaldehyde intrabiliary injections cause an inhibition of ursodeoxycholate-induced hypersecretion of bicarbonate, an increase in biliary glucose secretion, and selective structural alterations of biliary epithelial cells. These results suggest that formaldehyde retrograde biliary injection may be a useful model to study alterations of biliary epithelial function in vivo. (HEPATOLOGY 1996;24:1217-1223.) Bile formed by the hepatocytes (canalicular bile) is modified by the bile ductules and ducts on its way to the duodenum.1 Both absorptive and secretory processes have been described previously.1 The net effect is the addition of a bicarbonate-rich solution.2 Recently, the cellular and molecular mechanisms of secretion have been characterized: an adenosine 3*,5*-cyclic monophosphate–stimulated chloride channel, the cystic fibrosis transmembrane conductance regulator protein, has been identified on the apical membrane of rat and human biliary epithelial cells.3 The channel allows the extrusion of chloride ions that probably stimulate bicarbonate secretion through activation of a chloride/bicarbonate exchanger.4
Abbreviations: UDCA, ursodeoxycholic acid; AST, aspartate transaminase; LDH, lactate dehydrogenase. From the 1Unite´ de Recherches de Physiopathologie He´patique (INSERM Unite´ 24 and Association Claude Bernard) and Service d’He´patologie, Hoˆpital Beaujon, Clichy, France; and 2Unite´ de Recherches Structure et Fonctions des Cellules He´patiques (INSERM Unite´ 327), Faculte´ de Me´decine Xavier Bichat, Paris, France. Received December 8, 1994; accepted June 27, 1996. Address reprint requests to: Serge Erlinger, M.D., INSERM Unite´ 24, Hoˆpital Beaujon, 100 Bd du Ge´ne´ral Leclerc F-92118 Clichy Cedex, France. Copyright q 1996 by the American Association for the Study of Liver Diseases. 0270-9139/96/2405-0039$3.00/0
Hepa 0034
/
5P16$$$661
10-09-96 15:03:25
SERGE ERLINGER1
Biliary channels may also play a role in ursodeoxycholateinduced hypercholeresis.5-7 The most widely accepted hypothesis to account for bicarbonate-rich hypercholeresis induced by ursodeoxycholate is the so-called cholehepatic cycle,8 which implies passive absorption of protonated ursodeoxycholic acid (UDCA) through the biliary epithelial cells. Finally, biliary ducts play a role in the reabsorption of a number of organic compounds, including amino acids9 and glucose.10 Alteration of these functions are likely to occur in diseases affecting the biliary epithelium, such as primary biliary cirrhosis or primary sclerosing cholangitis. In this study, our purpose was to examine in vivo the functional consequences of an alteration of the biliary epithelium by a retrograde intrabiliary injection of formaldehyde. This agent has been shown to induce a secondary cholangitis in rats,11 dogs,12 and humans.13 We examined the immediate consequences of intrabiliary injections of formaldehyde on ursodeoxycholate-induced hypercholeresis and on biliary glucose secretion. We showed that ursodeoxycholate-induced hypercholeresis was decreased but not suppressed and that glucose biliary secretion was considerably increased. Hepatocyte excretory function, as assessed by biliary secretion of bile acids, was not affected. Thus, retrograde biliary injection of formaldehyde may be a useful model to study biliary function in vivo. MATERIALS AND METHODS Chemicals. UDCA, tauro-UDCA, and ethylcarbamate were pur-
chased from Sigma Chemical Co. (St. Louis, MO). Pentobarbital was purchased from Sanofi (Paris, France), and formaldehyde, 1-propanol, propionic acid, and isoamyl acetate were purchased from Merck (Darmstadt, Germany). Experimental Procedures. Male Sprague-Dawley rats (CD strain; Charles River Breeding Laboratories, St. Aubin-les-Elbeuf, France), weighing 243 { 20 g (mean { SD), were used. They were anesthetized intraperitoneally with pentobarbital (5 mg/100 g body wt). Rectal temperature was monitored and maintained at 387C { 0.57C on a heating table. Tracheostomy was performed. A jugular vein was cannulated with a polyethylene catheter no. 3 (ID, 0.58 mm; Biotrol, Paris, France), and the common bile duct was cannulated with a no. 1 catheter (ID, 0.3 mm; Biotrol). Bile was collected immediately after cannulation during three periods of 10 minutes. The mean of the two last periods was taken as basal values for bile flow. Then, a retrograde intrabiliary injection14 of 10% formaldehyde was performed in a volume of 40 mL/100 g body wt, corresponding to the volume of the biliary tree,15 and 10 mL for the cannula dead space. The bile duct cannula was occluded for 30 minutes. Control rats were administered 0.15 mol/L NaCl instead of formaldehyde. After 30 minutes, the cannula was opened, and bile flow was collected during two periods of 15 minutes to obtain a stable bile flow. Then bile was collected every 10 minutes for 90 minutes. In two other groups, rats were treated as described previously with 10% formaldehyde or NaCl and were administered an infusion of UDCA or tauro-UDCA at the rate of 800 nmolrmin01r100 g body wt01 for 90 minutes after the 30 minutes of bile flow stabilization. The mean of the two last periods was taken as UDCA values. Two additional groups of rats were studied to measure the bile/ plasma concentration ratio of sucrose. After ligation of renal pedicles, 5 mCi of [3H]sucrose (Amersham Radiochemicals, Amersham, England) was injected into the penile vein. After a period of equilibration of 60 minutes, the carotid artery and the common bile duct were
1217
AID
AND
hpta
WBS: Hepatology
1218
DUMONT ET AL.
HEPATOLOGY November 1996
FIG. 1. Experimental design.
cannulated. Bile samples were collected during 10-minute periods for 30 minutes. Thereafter, a retrograde intrabiliary injection of 10% formaldehyde (n Å 6) or NaCl (n Å 6) was performed, and the catheter was occluded for 30 minutes. Then the cannula was opened, and bile was collected for 30 minutes. Aliquots of 200 mL of arterial blood were taken at the midpoint of bile collection periods before and 60 minutes after the reopening of the bile duct cannula. The experimental design is shown in Fig. 1. Four adult Sprague-Dawley rats were prepared specifically for morphological investigation. Two rats were administered a retrograde intrabiliary injection of 10% formaldehyde, whereas two other rats were administered 0.15 mol/L NaCl instead of formaldehyde and were used as controls. The experimental procedure for these four rats was identical to that followed for the other rats: the bile duct cannula was occluded for 30 minutes and then reopened. The four rats were killed 30 minutes after the reopening of the cannula. Analytical Methods. Bile acid concentration was measured by the enzymatic method using 3a-hydroxysteroid dehydrogenase.16 Biliary concentration of glucose was measured with a glucose oxidase method using a kit phenol-amino-4-antipyrine-peroxidase (bioMerieux, Marcy l’Etoile, France). Biliary concentration of HCO0 3 was measured with a carbon dioxide analyzer (Corning 965; Ciba Corning Diagnostics, Halstead, England). Biliary bile acids (tauroursodeoxycholate, glycoursodeoxycholate, and ursodeoxycholate) were separated by thin-layer chromatography.17 Bile was deposited on 0.25mm silica gel 60 F254 plates (Merck). The solvent system used was as follows: water/1-propanol/propionic acid/isoamyl acetate (3:4:6:6, vol/vol). The plates were sprayed with water, and the spots were eluted in tetrasodium diphosphate solution (0.1 mol/L). Bile acid concentration was measured in each eluate with 3a-hydroxysteroid dehydrogenase.18 Standards of ursodeoxycholate, tauroursodeoxycholate, and glycoursodeoxycholate were used. For measurement of 3H activity, aliquots of 50 mL of plasma or bile were put into a glass vial, and 10 mL of scintillation medium (ACS; Amersham Radiochemicals) was added. The activity was measured in a liquid scintillation spectrometer (Minaxi, Tricarb 4000 series; Packard, Meriden, CT) with automatic correction for quenching. Bile/plasma ratio of [3H]sucrose activity was calculated. Serum aspartate transaminase (AST) and lactate dehydrogenase (LDH) activities were determined by standard enzymatic methods (Sigma Diagnostic and Biotrol kits, respectively). Histological and Ultrastructural Examination. For each rat, a part of the common bile duct and two liver fragments were immediately taken after the animal was killed. Liver fragments were obtained in the hilar region and at the periphery of the organ. Bile duct and liver fragments were divided in two parts for histological and electron microscopic examination. The part designed for histological examination was fixed in Bouin’s fluid and embedded in paraffin. Five-micrometer-thick sections were stained with hematoxylin-eosin and examined on light microscopy. The part designed for electron microscopic examination was cut immediately into 1-mm-thick blocks, and the blocks were fixed in a 2.5% solution of glutaraldehyde buffered with 0.1 mol/L phosphate buffer, pH 7.4, for 2 hours at 47C. After washing in phosphate buffer, the blocks were postfixed in a 1% solution of osmium tetroxide buffered with veronal buffer, pH 7.2, at room temperature for 1 hour. Blocks were dehydrated by graded alcoholic solutions and embedded in epoxy resins. Semithin sections stained with toluidine blue were made on each block to localize the bile ducts. At least three bile duct–containing blocks were cut for the ultrastructural examination of the common bile duct
AID
Hepa 0034
/
5P16$$$662
10-09-96 15:03:25
FIG. 2. Effect of formaldehyde on spontaneous and ursodeoxycholate-induced bile flow in the rat. UDCA or tauro-UDCA were infused at a rate of 800 nmolrmin01r100 g01. Each point represents the mean { 1SD. ●, Formaldehyde plus UDCA; j, formaldehyde plus tauro-UDCA; m, formaldehyde; s, NaCl plus UDCA; and n, NaCl.
and of each liver fragment. Ultrathin sections stained with uranyl acetate and lead citrate were analyzed with a JEOL 1010 electron microscope. All animals received humane care in compliance with international recommendations. Statistical Analysis. Results are expressed as means { SD. Differences between mean values were assessed statistically using the Student’s t test. RESULTS Effect of Formaldehyde on Spontaneous and Ursodeoxycholate-Induced Choleresis. After retrograde injection of formal-
dehyde (n Å 7), spontaneous bile flow was significantly greater (11.1 { 1.1 mLrmin01r100 g01) than in rats with a retrograde injection of NaCl (n Å 6) (7.3 { 1.6 mLrmin01r100 g01; P õ .001). In contrast, during infusion of ursodeoxycholate, bile flow was similar in rats administered formaldehyde (n Å 6) and in rats administered NaCl (n Å 6) (19.7 { 4.0 and 20.4 { 4.0 mLrmin01r100 g01; NS) (Fig. 2). In rats administered formaldehyde, bile flow during infusion of tauroursodeoxycholate was significantly less (13.9 { 2.4 mLrmin01r100
FIG. 3. Effect of formaldehyde on spontaneous and ursodeoxycholate-induced bile acid output in the rat. UDCA or tauro-UDCA were infused at a rate of 800 nmolrmin01r100 g01. Each point represents the mean { 1SD. ●, Formaldehyde plus UDCA; j, formaldehyde plus tauro-UDCA; m, formaldehyde; s, NaCl plus UDCA; and n, NaCl.
hpta
WBS: Hepatology
HEPATOLOGY Vol. 24, No. 5, 1996
DUMONT ET AL.
FIG. 4. Effect of formaldehyde on spontaneous and ursodeoxycholate-induced bicarbonate output in the rat. UDCA or tauro-UDCA were infused at a rate of 800 nmolrmin01r100 g01. Each point represents the mean { 1SD. ●, Formaldehyde plus UDCA; j, formaldehyde plus tauro-UDCA; m, formaldehyde; s, NaCl plus UDCA; and n, NaCl.
g01) than that observed during ursodeoxycholate infusion (19.7 { 4.0 mLrmin01r100 g01; P õ .05). Bile salt output was similar in animals injected with NaCl or formaldehyde and infused with ursodeoxycholate or tauroursodeoxycholate (Fig. 3). Because it is known that hypercholeresis induced by ursodeoxycholate is associated with an increase in bicarbonate concentration and secretion into bile,5 we measured biliary bicarbonate concentration and output in rats injected with NaCl and rats administered formaldehyde. As expected, in rats administered NaCl, bicarbonate concentration and output increased significantly during ursodeoxycholate infusion (concentration, 47.3 { 9.2 against 30.8 { 3.9 mmolrL01; P õ .01; output, 990.5 { 377.6 against 227.8 { 64.4 nmolr min01r100 g01; P õ .001; increase in bicarbonate output, 762 nmolrmin01r100 g01 or 334%). Bicarbonate concentration and output also increased in animals injected with formaldehyde (concentration, 35.1 { 4.1 against 28.5 { 1.6 mmolrL01; P õ .01; output, 694.7 { 188.1 against 316.4 { 47.2 nmolr min01r100 g01; P õ .001). However, the increase in bicarbon-
1219
FIG. 6. Effect of formaldehyde (j) on biliary excretion of ursodeoxycholate and its conjugates in the rat. UDCA was infused at a rate of 800 nmolr min01r100 g01. The concentrations of UDCA, tauro-UDCA, and glyco-UDCA in bile (expressed in percentage of total bile acids) were measured by thinlayer chromatography at 40, 60, and 90 minutes after the beginning of UDCA infusion. Each column represents the mean { 1SD. h, NaCl.
ate output was significantly less (379 nmolrmin01r100 g01 or 120%) than that of animals injected with NaCl (P õ .001) (Fig. 4). Bicarbonate concentration and output were not modified by tauroursodeoxycholate (Fig. 4). Effect of Formaldehyde on Biliary Glucose Secretion. Biliary glucose concentration (10.1 { 3.2 against 0.9 { 0.1 mmolrL01; P õ .001) and output (110.8 { 31.5 against 6.3 { 5.0 nmolrmin01r100 g01; P õ .001) were significantly greater in rats injected with formaldehyde than in those injected with NaCl. Similarly, during infusion of ursodeoxycholate, glucose biliary concentration (8.1 { 1.9 against 1.3 { 0.5 mmolrL01; P õ .001) and output (160.1 { 55.7 against 26.2 { 8.1 nmolr min01r100 g01; P õ .001) were significantly greater in rats injected with formaldehyde than in those injected with NaCl (Fig. 5). Effect of Formaldehyde on Biliary Excretion of Ursodeoxycholate and its Conjugates. To test whether the inhibition of
hypercholeresis and biliary bicarbonate secretion in formaldehyde-injected rats was caused by an alteration of the putative cholehepatic cycling,8 we measured the biliary excretion of unconjugated ursodeoxycholate. The percentage of unconjugated ursodeoxycholate and that of glyco- and tauro-conjugated ursodeoxycholate were similar in formaldehyde-injected animals and NaCl-injected controls (Fig. 6). Effect of Formaldehyde on [3H]Sucrose Bile/Plasma Ratio.
Before retrograde injection, basal bile/plasma ratios of [3H]sucrose were similar in the two groups (0.20 { 0.05 and 0.16 { 0.04). After retrograde intrabiliary injection, the bile/ plasma ratio was significantly greater in formaldehyde-injected rats (0.51 { 0.09) than in NaCl-injected rats (0.22 { 0.03; P õ .001). Effect of Formaldehyde on Serum Enzyme Activities. There was no difference in basal values of AST and LDH activities
TABLE 1. Serum Activities of AST and LDH Before and After Retrograde Injection of Formaldehyde or NaCl Into the Biliary Tree Retrograde Biliary Injection NaCl (n Å 6)
FIG. 5. Effect of formaldehyde on biliary glucose secretion in the rat. UDCA or tauro-UDCA were infused at a rate of 800 nmolrmin01r100 g01. Each point represents the mean { 1SD. ●, Formaldehyde plus UDCA; j, formaldehyde plus tauro-UDCA; m, formaldehyde; s, NaCl plus UDCA; and n, NaCl.
AID
Hepa 0034
/
5P16$$$662
10-09-96 15:03:25
Formaldehyde (n Å 6)
Enzyme Activities
Before
After
Before
After
AST (U/L) LDH (U/L)
76 { 11 356 { 83
171 { 45 581 { 131
80 { 27 309 { 81
131 { 61 436 { 159
hpta
WBS: Hepatology
1220
DUMONT ET AL.
AID
Hepa 0034
HEPATOLOGY November 1996
/
5P16$$0034
10-09-96 15:03:25
hpta
WBS: Hepatology
HEPATOLOGY Vol. 24, No. 5, 1996
DUMONT ET AL.
1221
FIG. 7. Histological and ultrastructural appearance of biliary cells in (A, C, and E) control rats and (B, D, and F) formaldehyde-treated rats. In control rats, note the normal structure of (A) the biliary cells of the common bile duct and of (C and E) a bile duct localized in a hilar portal space of the liver. The biliary epithelium [e] is composed of (A) cylindric or (C and E) cubic cells regularly arranged around the lumen [L] of the bile duct. (E) At the ultrastructural level, few organelles are visible in the cytoplasm of the cells. (B, D, and F) In formaldehyde-treated rats, the biliary epithelium [e] of the common bile duct is (B) separated from the surrounding connective tissue in some places. (B and D) Biliary cells are shrunken, and their nuclei and cytoplasm are difficult to recognize. At the ultrastructural level, note the (F) irregular shape of biliary cell nuclei, the absence of microvilli in front of the lumen [L], and the electrondense appearance of the cytoplasm (original magnification: A and B, 1501; C and D, 9001; E, 6,0001; and F, 7,0001). b
between the two groups. After retrograde injection of formaldehyde or NaCl, the serum activities of AST and LDH were greater than the basal activities. However, there was no significant difference between formaldehyde- and NaCl-injected animals (Table 1). Light Microscopy. In the control rats administered saline, the histological appearance of the biliary epithelial cells was normal either in the common bile duct (Fig. 7A) or in the portal bile ducts (Fig. 7C) localized in the hepatic hilar region. No alteration of these cells was observed in the bile ducts of the portal spaces visible in the peripheral regions of the liver. No inflammatory cells were present around the biliary ducts, whatever their localization. Hepatocytes were normal in the two regions of the liver examined. In the rats administered formaldehyde, the biliary epithelium was deeply altered. In the common bile duct, it was separated from the surrounding connective tissue by more or less large spaces (Fig. 7B). Moreover, the biliary cells were so shrunken that it was difficult to recognize their nucleus and cytoplasm. Similar changes were observed in the biliary ducts of the portal spaces localized either in the hepatic hilar regions (Fig. 7D) or at the periphery of the liver. However, alterations of bile duct cells were not visible in all of the examined portal spaces. In particular, some bile ducts of the peripheral portal spaces were normal or almost normal. Whatever the localization of the bile ducts, no inflammatory cells were observed around the biliary cells (Figs. 7B and D). In the liver, hepatocytes were normal around the portal spaces (Fig. 7D). Electron Microscopy. No alteration of the biliary cells was observed in control rats administered saline. A typical aspect of a bile duct from a hilar portal space is shown in Fig. 7E. Biliary cells with a prominent nucleus and a scanty cytoplasm were regularly arranged along the lumen of the bile ducts. At a greater magnification (Fig. 8A), intact tight junctions were visible. Microvilli were visible at the apical plasma membrane domain of the cells. A continuous basement membrane surrounded the biliary cells. No inflammatory cells were present in the connective tissue. The ultrastructural appearance of periportal hepatocytes was normal, and intact tight junctions (Fig. 8C) were observed at the level of bile canaliculi. In rats administered formaldehyde, the ultrastructural appearance of biliary cells was very often different from that observed in control rats. A typical aspect of a bile duct from a hilar portal space is shown in Fig. 7F. The nucleus of the biliary cells was very altered with an irregular size and profound invaginations. The cytoplasm was electron dense and shrunken. Cytoplasmic organelles were difficult to recognize. Microvilli were absent at the apical plasma membrane domain. At a greater magnification (Fig. 8B), tight junctions were unaltered. Similar ultrastructural aspects were observed in the biliary cells from the other parts of the bile ducts. However, some bile ducts were normal, in particular those localized in peripheral portal spaces. Finally, the ultrastructural appearance of periportal hepatocytes was normal, and intact tight junctions were observed around bile canaliculi (Fig. 8D). As in control rats, no inflammatory cells were visible around the bile ducts, whatever their localization in the biliary tract or in the liver.
AID
Hepa 0034
/
5P16$$$662
10-09-96 15:03:25
DISCUSSION
In these experiments, we attempted to impair selectively the function of the biliary epithelium by a retrograde intrabiliary injection of formaldehyde in rats in vivo. The first observation in this study was a significant inhibition of ursodeoxycholate-induced hypersecretion of bicarbonate in the rat. In this animal, ursodeoxycholate, when administered in the unconjugated form, induces a greater bile flow than tauroursodeoxycholate or taurocholate.5,6 This hypercholeresis is associated with an increase in biliary bicarbonate concentration and secretion.5,6 This phenomenon has been accounted for by the secretion of ursodeoxycholate in the unconjugated form in canalicular bile followed by its protonation (with generation of bicarbonate) and passive reabsorption by the biliary epithelial cells8 and its return to the liver by the peribiliary plexuses. At each of these cholehepatic cycles, a bicarbonate ion is generated, explaining the increase in biliary bicarbonate concentration and secretion. In rats injected with formaldehyde, we observed a significant inhibition of ursodeoxycholate-induced bicarbonate output (Fig. 4). However, passive absorption of ursodeoxycholate was not affected because the proportion of unconjugated ursodeoxycholate in the final bile was unchanged (Fig. 6). If cholehepatic cycling had been interrupted, we would have expected an increase in unconjugated ursodeoxycholate in the bile. The decrease in bicarbonate generation (and, consequently, of bicarbonate-induced bile flow) may be explained by an inhibition of luminal carbonic anhydrase by formaldehyde. The protonation of ursodeoxycholate is caused by subtraction of H/ from carbonic acid, thereby producing an increase in biliary HCO0 3 concentration. Because the CO2 -H2CO3 buffer is an open system and CO2 freely crosses biological membranes, H2CO3 is formed by luminal carbonic anhydrase.19 Inhibition of this enzyme may lead to decreased H2CO3 generation and availability and, thus, to decreased HCO0 3 formation. Two pieces of evidence support this interpretation. First, previous studies from this and other laboratories have shown that acetazolamide, a known inhibitor of carbonic anhydrase, also inhibited the stimulation of bicarbonate secretion induced both by ursodeoxycholate and secretin.20,21 Second, glutaraldehyde, which has chemical similarities with formaldehyde, is known to inhibit carbonic anhydrase.22 Studies of isolated biliary epithelial cells may help to test this hypothesis. The second observation in this study is a marked increase in glucose biliary secretion in formaldehyde-injected rats. In the normal state, biliary glucose concentration is remarkably low. This has been explained by a passive glucose entry into canalicular bile followed by an active glucose reabsorption by the biliary epithelial cells.23 This reabsorption seems to be mediated by two luminal glucose transport systems: a sodium-dependent and a sodium-independent system.23 Our results are consistent with an inhibition of these transport systems by formaldehyde, resulting in increased glucose output. In turn, this increased glucose output should result in an increased bile flow by an osmotic mechanism. Our observations are again consistent with this hypothesis. In rats injected with formaldehyde, basal bile flow was increased significantly. Glucose output was also increased. We propose that the most likely explanation for this increase in bile flow is an osmotic effect of glucose. Similarly, when UDCA was
hpta
WBS: Hepatology
1222
DUMONT ET AL.
HEPATOLOGY November 1996
FIG. 8. Ultrastructural appearance of tight junctions [TJ] of biliary cells and hepatocytes in (A and C) control rats and (B and D) formaldehyde-treated rats. The junctions appear morphologically normal in the two groups of rats, not only between the biliary cells (observed here in a bile duct from a hepatic hilar portal space) but also in periportal hepatocytes (A, 28,0001; B, 16,0001; C, 34,5001; and D, 32,0001). N, nucleus; H, hepatocyte; BC, bile canaliculus; mv, microvilli.
infused in rats injected with formaldehyde, ursodeoxycholate-induced HCO0 3 secretion was significantly less than that in NaCl-injected rats, but bile flow was virtually identical. The most likely explanation is that glucose was the osmotically active compound responsible for the increase in basal bile flow and the maintenance of bile flow under ursodeoxycholate infusion. Assuming an osmotic activity of 3-10 mL/ mmol23 and taking into account a biliary secretion of glucose of 0.11 mmolrmin01r100 g01, the glucose-induced bile flow should be 0.33-1.1 mLrmin01r100 g01. The observed increase in bile flow (11 mLrmin01r100 g01 in formaldehyde-injected rats vs. 7.3 mLrmin01r100 g01 in NaCl-injected controls) is far greater than the calculated value. This was also the case in the experiments by Lira et al.,23 where the amount of fluid reabsorbed in the bile ducts exceeded that predicted from the amount of glucose reabsorbed. We have to postulate, as did Lira et al., that reabsorption of other substrates (such as amino acids or inorganic electrolytes) is also inhibited by
AID
Hepa 0034
/
5P16$$$663
10-09-96 15:03:25
formaldehyde. A possible substrate could be glutathione: glutathione is secreted into bile by the hepatocytes and cleaved in the biliary lumen into several peptides by g-glutamyl transferase.24 These peptides are reabsorbed in the biliary tree. g-Glutamyl transferase is inhibited by incubation with formaldehyde (data not shown); this would result in an increased biliary secretion of glutathione. An alternative explanation for the increase in biliary glucose concentration and secretion may be that formaldehyde increased the permeability of the biliary epithelium to glucose. Because glucose concentration in plasma is greater than in bile, an increase in biliary permeability could facilitate the movement of glucose from plasma to bile. To test this possibility, we estimated the permeability between blood and bile by measuring the bile/plasma concentration ratio of sucrose.25 We found a significant increase in the bile/plasma ratio after formaldehyde injection: the bile/plasma ratio was greater in rats administered formaldehyde than in the NaCl-
hpta
WBS: Hepatology
HEPATOLOGY Vol. 24, No. 5, 1996
DUMONT ET AL.
injected controls. This indicates that the permeability to sucrose was increased. Therefore, it is likely that the permeability to glucose was also increased. Theoretically, this increase in permeability may be caused by an alteration: of the hepatocytes; of the tight junctions around bile canaliculi; of the biliary epithelial cells; or of tight junctions between biliary cells. Therefore, we performed a histological and ultrastructural study. The results indicated that the hepatocytes and tight junctions around bile canaliculi were always normal. There were alterations of the intrahepatic biliary epithelial cells, particularly of their apical membrane. However, tight junctions between these cells appeared to be morphologically intact. The most striking alterations were located on the extrahepatic bile ducts. The extrahepatic bile ducts appear to be the most likely site of the increased permeability. Finally, we present evidence that the hepatocytes were functionally and morphologically intact after formaldehyde injection. First, bile acid secretion after ursodeoxycholate and tauroursodeoxycholate infusions was normal. This indicates a normal hepatocyte excretory function. Secondly, hepatocyte enzymes (AST and LDH activities) were only moderately elevated after intrabiliary injection and not significantly different between formaldehyde- and NaCl-injected animals. Thirdly, the ultrastructural appearance of hepatocytes and hepatocyte tight junctions was always normal. Toxicity of formaldehyde on the liver has been reported in the literature,26,27 mostly on the isolated perfused rat liver and isolated rat hepatocytes. This was apparently not the case in our experimental system, perhaps because formaldehyde was injected in the biliary tree rather than in the circulation or directly in contact with hepatocytes. None of the ultrastructural lesions previously reported (mitochondrial damage or alterations of the rough endoplasmic reticulum) was observed in this study. In summary, formaldehyde intrabiliary injection resulted in an inhibition of ursodeoxycholate-induced bicarbonate secretion in rats and in a marked increase in biliary glucose. These observations are consistent with a profound and selective alteration of biliary epithelial function by formaldehyde. The alteration was selective because the excretory function and morphology of hepatocytes were normal. Alteration of biliary epithelial function was probably caused by both inhibition of transport systems of biliary epithelial cells and destruction of cells of the extrahepatic bile ducts, resulting in increased biliary permeability. Intrabiliary injection of formaldehyde may be a useful model to study alterations of biliary epithelial function in vivo.
4. 5. 6. 7.
8.
9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24.
REFERENCES 1. Erlinger S. Secretion of bile. In: Schiff E, Schiff L, eds. Diseases of the liver. Philadelphia: Lippincott, 1993:85-107. 2. Tarsetti F, Lenzen R, Salvi R, Schuler E, Dembitzer R, Tavoloni N. Biology and pathobiology of intrahepatic biliary epithelium. In: Tavoloni N, Berk PD, eds. Hepatic transport and bile secretion: physiology and pathophysiology. New York: Raven, 1993:619-635. 3. Cohn JA, Strong TV, Picciotto MR, Nairn AC, Collins FS, Fitz JG. Localiza-
AID
Hepa 0034
/
5P16$$$663
10-09-96 15:03:25
25. 26. 27.
1223
tion of the cystic fibrosis transmembrane conductance regulator in human bile duct epithelial cells. Gastroenterology 1993;105:1857-1864. Fitz JG, Basavappa S, McGill J, Melhus O, Cohn JA. Regulation of membrane chloride current in rat bile duct epithelial cells. J Clin Invest 1993; 91:319-328. Dumont M, Erlinger S, Uchman S. Hypercholeresis induced by ursodeoxycholic acid and 7-ketolithocholic acid in the rat: possible role of bicarbonate transport. Gastroenterology 1980;79:82-89. Kitani K, Kanai S. Effect of ursodeoxycholate on the bile flow in the rat. Life Sci 1982;31:1973-1985. Gurantz D, Schteingart CD, Hagey LR, Steinbach JH, Grotmol T, Hofmann AF. Hypercholeresis induced by unconjugated bile acid infusion correlates with recovery in bile of unconjugated bile acids. HEPATOLOGY 1991;13:540-550. Yoon YB, Hagey LR, Hofmann AF, Gurantz D, Michelotti EL, Steinbech JH. Effect of side-chain shortening on the physiological properties of bile acids: hepatic transport and effect on biliary secretion of 23-nor-ursodeoxycholate in rodents. Gastroenterology 1986;90:837-852. Ballatori N, Jacob R, Barrett C, Boyer JL. Biliary catabolism of glutathione and differential reabsorption of its amino acid constituents. Am J Physiol 1988;254:G1-G7. Guzelian P, Boyer J. Glucose reabsorption from bile, evidence for biliohepatic circulation. J Clin Invest 1974;53:526-535. Houry S, Languille O, Huguier M, Benhamou JP, Belghiti J, Msika S. Sclerosing cholangitis induced by formaldehyde solution injected into the biliary tree of rats. Arch Surg 1990;125:1059-1061. Grimaud JC, Sastre B, Orsoni P, Monges G, Treffot MJ, Monges A, Michotey G. Le roˆle du formol dans la survenue de le´sions scle´rosantes des voies biliaires. J Chir (Paris) 1984;121:163-169. Belghiti J, Benhamou JP, Houry S, Grenier P, Huguier M, Fe´ke´te´ F. Caustic sclerosing cholangitis. A complication of the surgical treatment of hydatid disease of the liver. Arch Surg 1986;121:1162-1165. Farges O, Corbic M, Dumont M, Maurice M, Erlinger S. Permeability of the rat biliary tree to ursodeoxycholic acid. Am J Physiol 1989;256:G653G660. Barber-Riley G. Measurement of capacity of biliary tree in rats. Am J Physiol 1963;205:1122-1126. Berthelot P, Erlinger S, Dhumeaux D, Preaux AM. Mechanism of phenobarbital-induced hypercholeresis in the rat. Am J Physiol 1970;219:809813. Hofmann AF. Thin-layer adsorption chromatography of free and conjugated bile acids on silicic acid. J Lipid Res 1962;3:127-128. Turnberg LA, Anthony-Mote A. The quantitative determination of bile salts in bile using thin-layer chromatography and 3a-hydroxysteroid dehydrogenase. Clin Chim Acta 1969;24:253-259. Strazzabosco M, Sakisaka S, Hayakawa T, Boyer J. Effect of UDCA on intracellular and biliary pH in isolated rat hepatocyte couplets and perfused livers. Am J Physiol 1991;260:G58-G69. Garcia-Marin JJ, Dumont M, Corbic M, de Couet G, Erlinger S. Effect of acid-base balance and acetazolamide on ursodeoxycholate-induced biliary bicarbonate secretion. Am J Physiol 1985;248:G20-G27. Buanes T, Grotmol T, Landsverk T, Ridderstra˚le Y, Ræder MG. Importance of carbonic anhydrase for canalicular and ductular choleresis in the pig. Acta Physiol Scand 1988;133:535-544. Buanes T, Grotmol T, Landsverk T, Ridderstra˚le Y, Ræder MG. Histochemical localization of carbonic anhydrase in the pig’s exocrine pancreas. Acta Physiol Scand 1986;128:437-444. Lira M, Schteingart CD, Steinbach JH, Lambert K, McRoberts JA, Hofmann AF. Sugar absorption by the biliary ductular epithelium of the rat: evidence for two transport systems. Gastroenterology 1992;102:563-571. Ballatori N, Jacob R, Barrett C, Boyer JL. Biliary catabolism of glutathione and differential reabsorption of its amino acid constituents. Am J Physiol 1988;254:G1-G7. Forker EL. The effect of estrogen on bile formation in the rat. J Clin Invest 1969;48:654-663. Ku RH, Billings RE. Relationships between formaldehyde metabolism and toxicity and gluthatione concentrations in isolated rat hepatocytes. Chem Biol Interact 1984;51:25-36. Strubelt O, Younes M, Pentz R. Mechanistic study on formaldehyde-induced hepatotoxicity. J Toxicol Environ Health 1989;27:351-366.
hpta
WBS: Hepatology