life Sciences. Vd.63. No.22.pp. I%51974, 1998 Copyright 0 1998 Ekmier Science Inc.

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ELSEVIER PI1 sa0X3205(98)00474-3 cn?A-3m/98 519.00 + .a0

ENHANCED BILE FORMATION INDUCED BY EXPERIMENTAL DICROCOELIOSIS IN THE HAMSTER

S. Sanchez-Campos, M. J. Ttion, P. Gonzalez, *J. J. G. Marin and J. Gonzalez-Gallego

Department of Physiology, Pharmacology and Toxicology, University of Leon, and *Department of Physiology and Pharmacology, University of Salamanca, Spain

(Received in final form September 14, 1998)

The purpose of this investigation was to determine the effects of experimental dicrocoeliosis on bile formation in the hamster. Studies were carried out at 120 days after infection with an oral dose of 40 metacercariae of Dicrocoelium dendriticum. A significaat elevation in bile flow (+20%) and in the biliary output of glutathione (+34%), bile acid (+59%), cholesterol (+108%), phospholipids (+99%) and alkaline phosphatase (+36%) was observed in the infected animals. The bile-to-plasma [14C] mannitol ratio increased to values greater than 1 and there was a reduced contribution (- 26%) of biliary tree to bile formation. Those data suggest that enhancement in choleresis had a canalicular origin. The presence of oxidative stress, evidenced by the increased oxidized’reduced glutathione ratio and TBARS concentrations, may contribute to the elevated glutathione efflux into bile. Enhancement in bile acid output was not due to qualitative or quantitative changes in bile acid metabolism, as indicated by the absence of significant modification in liver cholesterol 7a-hydroxylase activity and bile acid profile in bile. Increase in the ability of the canalicular membrane to export bile acids was not involved, since maximal secretion rate for exogenously administered taurocholate was decreased. When bile flow, bile acid and biliary lipid secretion was determined in colchicine-pretreated animals differences between control and infected animals were abolished, suggesting that stimulation of the transcytotic vesicle pathway plays an important role in the alteration of the biliary function caused by dicrocoeliosis.

Key Wordr: hamster, choleresis, bile acid, glutathione, cholesterol, phospholipid

Dicrocoelium dendriticum is a world wide distributed parasitic trematode which lives in the bile ducts of sheep, cattle and, occasionally, humans (1,2). Infective eggs develop in different species of land snails and the metacercariae reach infective stage in ants of the genus Formica. When ingested by the definitive host, their protective envelope is softened by the digestive juices and the young flukes migrate from the intestine via the ductus choledocus or via the portal blood

Correspondirzg author: J. Gonzalez-Gallego Ph.D., Departamento de Fisiologia, Farmacologia y Toxicologfa, IJniversidad de Leon, 24071 Leon, Spain. Tel 34 987 291258; Fax 34 987 291267; E- mail dftjgg@unileon.es

1964 Biliary Function in Experimental Dicrocoeliosis Vol. 63, No. 22,1998

system (3,4). In the first days of infection there is an angiectasis of the central veins and the portobiliary vessels. After the termination of the acute stage, a chronic inflammation of the biliary ducts with a marked proliferation of connective tissue develops (3). Apart from the mechanical irritation caused by the migrant young flukes, pathologic changes have been ascribed to the toxic effects of metabolic products released by the parasite (4).

The effects of other trematode infections, such as fasciolosis on the mechanisms of bile formation and liver detoxification systems have been extensively studied. Development of cholestasis, choleresis, changes in bile composition and alterations in liver phase I and phase II enzymes have been reported (5-7). Information on dicrocoeliosis, however, is very limited, probably because D. dendriticum generally does not cause severe pathological manifestations and the life cycle of the parasite is a complicated laboratory model for screening experiments. We have recently shown that experimental dicrocoeliosis produces an impairment of the capacity of the hamster liver for handling of drugs and xenobiotics (8) but there is a lack of information about the changes induced by this parasitism on the hepatobiliary function. The purpose of our study was to investigate the effects of experimental dicrocoeliosis on bile formation and biliary lipid secretion in the hamster.

Methods

Chemicals

Colchicine, 5P-cholestane, cholylglycine hydrolase, 5,5’-dithiobis-(2nitrobenzoic acid),

glutathione reductase, 3a-hydroxysteroid dehydrogenase, nicotinamide adenine dinucleotide, nicotinamide adenine dinucleotide phosphate, sodium taurocholate, cholic acid (CA), chenodeoxycholic acid (CDCA), deoxycholic acid (DCA) and lithocholic acid (LCA) were obtained from Sigma Chemical Co. (St Louis, US). Nordeoxycholic acid (Nor-DCA) was obtained from Calbiochem (San Diego, US). All bile acids were more than 95% pure by thin-layer chromatography. [i4C] mannitol was obtained from DuPont (Boston, US). All other reagents were the highest quality available commercially. Distilled deionized water was used throughout.

Animal Treatment Male Golden Syrian hamsters (Mesocricetus auratus) (Charles River, Barcelona, Spain), body

weight SO-120 g, were housed in cages in a temperature controlled room (22°C) with humidity ranging from 45% to 55% and a 12 hr-12 hr dark/light cycle. The animals had free access to food and water. Animals received, by gastric tubing, 40 metacercariae of Dicrocoelium dendriticum suspended in 0.154 M NaCl (8). The metacercariae used to infect the hamsters were recovered from the ant Formica rufibarbis (8,9). Parallel studies were carried out on uninfected control animals. Experiments were carried out 120 days after the infection, a period with histological and biochemical alterations characteristic of the chronic stage of the disease clearly established (8). All animals received human care as outlined in Guide to the Care and Use of Laboratory Animals (NIH Publication no. 80-23, revised 1985) which is routinely used at our laboratory.

Experimental Procedures Non-stimulated bile flow, composition of bile and biliary clearance of [14C] mannitol were

measured in both control and infected hamsters. The effect of endovenous infusion of taurocholate in these animals was also investigated. In a separate set of experiments the effect of pretreatment with colchicine, a microtubule inhibitor, on non-stimulated bile formation was examined.

Vol. 63, No. 22, 1998 Biliary Function in Experimental Dicrocoeliosis 1%5

All animals were anaesthetized with pentobarbital sodium (50 mg/kg body weight) given intraperitoneally and a median laparotomy was performed. The common bile duct, right carotid artery and internal jugular vein were cannulated with polyethylene tubing for collection of bile and blood samples, and for infusion of solutions, respectively. In those hamsters receiving [i4C] mannitol the renal pedicles were ligated. Body temperature was monitored with a digital rectal thermometer and kept between 37°C and 375°C with a heating lamp. All solutions for intravenous infusion were prepared in 0.154 M NaCl at 37°C. Bile was collected in preweighed vials for 1 hour in 20-min aliquots to establish basal bile flow, bile acid output, and biliary lipid composition. After the collection of these baseline samples of bile, both control and infected animals received either taurocholate or [t4C] mannitol infusion. Taurocholate (0.5 umol/lOO g/min, i.e., approximately 140 nmol/g liver/min) continuous infusion for 1 hour or [14C] mannitol(0.5 uCi/hr) infusion for two hours were performed using a peristaltic pump at 2 mlhr. Bile samples were collected in 20-min aliquots. Blood samples for [14C] mannitol radioactivity measurement were obtained from the carotid artery cannula after 70 min of i.v. infusion. Additional groups of control and infected hamsters were injected intraperitoneally with 0.2 mg/lOOg body wt of colchicine prepared in 0.154 M NaCl. Experiments were carried out three hours after injection (10).

All animals were killed after bile collection by exsanguination and the livers were rapidly removed and perfused through the hepatic veins with ice-cold 0.154 M NaCl to clear the liver of blood and frozen at -80°C. Plasma was obtained from blood samples by centrifugation. Bile and plasma samples were immediately stored at -40°C until required for analysis.

Analytical Procedures Bile flow was determined gravimetrically assuming a bile density of 1.0 g/ml. Total bile acids

in bile were measured with a 3a-hydroxysteroid dehydrogenase enzyme method (11). Amidated bile acids in bile samples were hydrolyzed enzymatically using cholylglycine hydrolase. Unconjugated bile acids were then extracted by liquid-solid extraction in C 18 cartridges. Methyl ester derivatives were prepared by reaction with ethereal diazomethane (12). Trimethylsilyl ether derivatives were prepared in pyridine-hexamethyldisilazane-trimethylchlorosilane 3:2:1 (by vol) overnight at room temperature (13). Gas chromatography-mass spectrometry (GC-MS) analyses were performed using a modification of the method described by Malavolti et al (14) on a gas chromatography connected to a mass spectrometer (HP 5890 series II and HP 5972, Hewlett- Packard, Madrid, Spain). A comparison with authentic standards was used to identify individual bile acids and measure their concentrations. When these were not available, identification was tentative based on the predicted fragmentation patterns for specific bile acid structures (15,16), and the amount of this bile acid was calculated as relative to the peak height response obtained for CA. Two internal standards were used to calculate the amount of bile acid lost during sample preparation (nordeoxycholic acid, a bile acid absent in hamster bile) and GC-MS analysis (5p- cholestane). Cholesterol concentration in bile was determined using cholesterol oxidase, cholesterol esterase and peroxidase (17). Phospholipids in bile were hydrolyzed by phospholipase D and the choline released was measured by the Trinder reaction (18,19). Alkaline phosphatase activity was determined by measuring the production of p-nitrophenol from p-nitrophenyl phosphate at 405 mn. A unit (U) of enzyme activity was defined as that amount that liberates 1 umol of p-nitrophenol per hour (20). Plasma and bile radioactivity derived to [K] mannitol infusion was measured in a liquid scintillation spectrometer (LS-1800-Beckman, Beckman Instruments, Madrid, Spain) using Ready-Safe cocktail, also from Beckman, as scintillant, obtaining the bile-to-plasma (B/P) ratio of radioactivity. The biliary clearance of [14C] mamtitol was calculated as the product of bile flow and the solute bile-to-plasma ratio (B/P). Biliary glutathione concentration was determined by an enzymatic method (21). Glutathione reduced and

1966 Biliary Function in Experimental Dicrocoeliosis Vol. 63, No. 22, 1998

oxidised analysis in liver was performed fluorimetrically by the method of Hissin and Hilf (22). The activity of cytosolic gamma-glutamyl-cysteinyl synthetase was measured as described by Kretzschmar et al (23). Thiobarbituric acid reactive substances (TBARS), a marker of lipid peroxidation, were determined in liver homogenates according to Ohkawa et al (24). Liver microsomal fractions were prepared by differential centrifugation of a 20% homogenate using established conditions (25). Protein concentration was measured according to Lowry et al (26) using crystallized bovine plasma albumin as a standard. The specific activity of cholesterol 7a- hydroxylase was determined in microsomes using an HPLC assay procedure as previously described (27). Small portions of the liver for light microscopy were fixed in 4% formol saline, dehydrated and embedded in paraffin. Sections 3-4 mm thick were obtained and were stained with Masson-Goldner trichrome.

Statistical Methods The peak of bile acid secretion rate during taurocholate administration was determined as the

mean of the three highest consecutive values of bile acid output. Results are expressed as mean*SEM. The data were compared by ANOVA, when the analysis indicated the presence of a significant difference the means were compared by t test for paired or unpaired values, and the Bonferroni method of multiple range testing as appropriate. Analyses were run on the SPPS for Windows statistical package (SPSS Federal Systems; Chicago, US).

Results

The presence of subclinical dicrocoeliosis was confirmed by the recovery of eggs in faeces of animals from 8 weeks after infestation (mean faecal egg count 568+62). Number of adult flukes recovered in the liver and bile ducts was 1954. At 120 days post infection there was no difference in body weight or liver weight between control and parasited hamsters (body weight: 11714 g vs 116*2 g; liver weight: 3.6hO.l g vs 3.5kO.l g).

Fig. 1 Intrahepatic bile duct at 120 days post infection. Duct is dilated and wall shows fibrous thickening with some infiltration by chronic inflammatory cells. Masson Goldner trichrome. Ob. x 6.3.

Vol. 63, No. 212, 1998 Biliary Function in Experimental Dicrocoeliosis 1%7

Histological examination revealed the existence of dilated intrahepatic bile ducts lined by proliferative epithelium. Wails showed fibrous thickening with some infiltration by chronic inflammatory cells (Figure 1). Portal tracts were occasionally infiltrated by lymphocytes and macrophages. A dilatation of sinusoids and a discrete increase-in the number of Kupffer cells was also observed

When compared to control animals significantly increased values in bile flow (+20%) and bile output of bile acids (+59%), phospholipid (+99%), cholesterol (+ lo@??), alkaline phosphatase (+36%) and glutathione (+34%) were found in infected hamsters (Table 1).

TABLE 1

Effects of Experimental Dicrocoeliosis on Basal Bile Flow and Composition

Control Infected

Bile flow (ph’g liver/min) Cholesterol (nmollg liver/min) Phospholipid (mnol/g liver/min) Glutathione (nmol/g liverimin) Bicarbonate (nmol/g liver/min) Alkaline phosphatase &U/g liver/min) Bile acids

Total bile acids (nmol/g liver/min) Profile (?h)

Major bile acids CA CDCA DCA LCA Minor bile acids

3a, 12p dihydroxy-5P-cholanoic acid 3a,7a,12l3 trihydroxy-5b-cholanoic acid 3oxo,12a hydroxy-5b-cholanoic acid

7ox.o,3a,12a dihydroxy-5l3-cholanoic acid 120x0, 3a,7a dihydroxy-5P-cholanoic acid

Others Ratios

CA+DCA/CDCA+LCA CA+CDCA/DCA+LCA

1.39*0.06 0.79*0.07 1.44kO.20 3.07*0.3 1 74.2k9.7 197k29

22.1h2.2

75.2h3.7 17.1+3.4 5.4kO.5 0.59hO.09

0.29*0.04

0.19kO.04 0.22kO.06 0.91kO.28

0.03*0.03 0.03kO.02

5.31*1.38 15.8k1.7

1.67*0.06* 1.64&0.19* 2X7+0.44* 4.1 l&0.46* 68.9h4.6 267+125*

35.1*3.5*

68.5k4.0 21.3i4.4 7.2&l .O 0.52ztO.10

0.4010.08

0.3&0.03 0.1 l&O.04 1.29kO.41 0.29*0.11* 0.07*0.02

4.lti1.01 12.2&l .6*

Values are :means+SEM (n=lO in both groups). *p<O.O5 significantly different from control.

Activity of cholesterol 7a_hydroxylase, the rate limiting enzyme in bile acid synthesis, was not significantly changed in infected animals (15.2kO.9 vs 14.8+1.0 nmol/mg protein/min in the controls). Cbmges in the proportion of the different molecular species of bile acids were investigated using GC-MS (Table 1). No marked alteration in bile acid profile in bile was found, but small changes, such as the reduction in primary- to secondary-bile acid ratio and the increase

1968 Biliary Fundion in Experimental Dicrocoeliosis Vol. 63, No. 22, 1998

in the proportion of oxo-bile acids appeared. Total bile acid calculated from GC-MS revealed that

less than 10% of the amount of these molecules present in bile was not detected by 3a- hydroxysteroid dehydrogenase method.

Liver ability to handle a bile acid load was studied by intravenous infusion of sodium taurocholate (0.5 pmol/lOO g/min) Bile acid secretion peak was significantly lower in infected than in control animals (-21%). Cholesterol, phospholipid and alkaline phosphatase secretion did not significantly differ between control and parasited animals (Table 2).

TABLE 2

Peak of Bile Flow and Bile Acid, Phospholipid, Cholesterol and Phosphatase Alkaline Secretion Rate during Taurocholate Administration to Control and Infected Hamsters

Control Infected

Bile flow (pmol/g liver/min) 2.9lztO.23 2.34+0.13* Bile acid (nmol/g liver/min) 178513 141&11* Cholesterol (mnoVg liver/min) 1.83hO.34 2.2WO.27 Phospholipid (nmol/g liver/min) 5.9951.29 6.54hO.40 Alkaline phosphatase @U/g liver/min) 426&47 474*39

Values are mea&&EM (n=15 in both groups). *p<O.O5 significantly different from control

*

Fig. 2 1” Effects of experimental infection on [“Cl mannitol clearance and bile/plasma ratio. Values are mean&SEM (n=lO animals in both groups). *p<O.O5 significantly different from control.

To obtain more information on the mechanisms of enhanced bile formation, [14C] mannitol was used as a marker of hepatocytic bile flow. Both [14C] mannitol clearance and solute bile/plasma ratio were significantly increased in parasited animals (+32% and +49%, respectively) (Fig. 2).

Vol. 63, No. :!2, 1998 Biliary Function in Experimental Dicrocoeliosis 1%9

Duct&r bile flow, obtained from the difference between [14C] mtitol clearance and mean bile flow, was reduced from 0.43&O. 10 pi/g liver/min in control animals to 0.3aO.07 pi/g liver/min.

Total liver glutathione concentrations were not significantly modified by the parasitosis (7.26~tO.19 vs 7.2OkO.24 mmoVg liver in the controls), but the oxidised/reduced glutathione ratio (GSSG/GSH) was significantly increased (+145%) (Fig. 3). TBARS concentration and gamma glutamylcysteinyl synthetase activity were significantly higher in the liver of infected animals (+84% and +20%, respectively) (Fig. 3).

I 0 Control q Infected

0.03 1 180, -

Fig. 3 Liver GSSG/GSH ratio, TBARS concentration and gamma-glutamylcysteinyl synthei.ase (yGCS) activity in control and infected animals. Values are meantiSEM (n=lO animals in both groups). * ~~0.05 significantly different from control.

To investigate whether enhanced biliary lipid secretion caused by dicrocoeliosis was related to alterations in the transcytotic vesicle pathway, colchicine (0.2 mg/lOO g body wt) was administered three hours before bile collection. Colchicine had no significant effect on the biliary secretion of bile acids, phospholipid or cholesterol in control animals (Fig. 4). However, a significant decrease in the secretion of all biliary lipid species was found when colchicine-treated infected animals were compared to untreated infected ones. No colchicine-treated control and infected animals was found in either and phospholipid secretion (Fig. 4).

significant difference between bile flow, bile acid, cholesterol

Discussion

Bile is a fluid secreted by the hepatocytes into the bile canaliculi and modified in the bile ductules or ducts. Active secretion of bile acids and their conjugates accounts for at least 40-50% of canalicular bile flow while canalicular bile acid-independent secretion is driven by organic compounds different from bile acids and the bile ducts and ductules secrete a bicarbonate-rich fluid stimulated by secretin (28). Measurement of the biliary clearance of inert carbohydrates, such as erythritol and mannitol, has been extensively used to differentiate canalicular and ductular

1970 Biliary Function in Experimental Dicrocoeliosis Vol. 63, No. 22,1998

components of bile flow. Although the validity of these approaches has been questioned on the basis of evidence that these solutes permeate also the biliary epithelium (29), this route contributes a maximum of only 10% of their total biliary entry (30). Results obtained in the present work demonstrate an increased mannitol clearance, suggesting that enhanced bile flow induced by the Dicrocoehm denditricum infection in hamsters mainly occurs at the canalicus.

It is well known that canalicular bile can be modified during its transit through the biliary tree (31). Water reabsorption is assumed to be the cause for the finding that bile-to-plasma ratio of inert carbohydrate, not actively transported into bile (32), is greater than 1 in several species, such as dogs or monkeys. Our results indicate that an increase in bile-to-plasma mannitol ratio above unity together with a marked (-26%) reduction in the contribution of biliary tree to bile formation occur in infected hamsters. Bile duct proliferation and fibrous thickening of epithelial wall are probably involved in these changes.

Fig. 4

E H Control+colchlcme

-

l-

-l-

Effects of colchicine administration on bile flow and biliary lipid secretion in control and infected animals. Values are means *SEM (n=5 animals in both groups). * ~~0.05 significantly different from control. # ~~0.05 significantly different from infected.

Vol. 63, No. 22, 1998 Biliary Function in Experimental Dicrocdiosis 1971

Different investigations support that, in addition to bicarbonate, actively secreted glutathione is the principal driving force in canalicular bile acid-independent bile flow (33). Evidence for the role of glulathione in bile formation has been provided by inhibiting degradation with acivicin (34) and by studies in the adult mutant Groningen Yellow (35). Agents that increase or decrease glutathiont: secretion in the isolated perfused rat liver, produce similar changes in bile flow, with glutathione linearly related to bile flow (36). In the present investigation on infected hamsters, biliary bicarbonate secretion was not modified, but glutathione secretion into bile was markedly increased. This support the hypothesis that increased biliary secretion of glutathione, but not bicarbonate, contributes to the enhancement in bile flow observed in animals with dicrocoeliosis.

Glutathione plays a key role in the maintenance of the cell redox status and may be consumed by conjugation reactions with metabolites of xenobiotics or reduced glutathione may be oxidised to the disulfide form (GSSG) that is excreted preferentially into bile (37). Different studies have shown thal. biliary efflux of GSSG is induced by perfusion of hydroperoxides or drugs that produce H202 in situ (38). Dicrocoeliosis coincided with the development of oxidative stress, as shown by the increase in liver TBARS and the increased GSSG to reduced glutathione ratio. and this could contribute to explain the rise in biliary glutathione levels. The reason for the presence of oxidative alterations is unknown at present but toxins released by the parasite could be involved (4). In parasited animals, the activity of gamma glutamylcysteinyl synthetase, a limiting enzyme in glutathione synthesis, was also significantly increased. Induction of this enzyme associated to overexpression of the canalicular transporter MRPl, responsible for transport of glutathione conjugate: and GSSG, has been previously reported (39) and it has been shown that both appear to bc corregulated (40).

A major finding of the present work is that bile acid output and hence the bile acid-dependent bile flow was markedly increased in infected animals. Cholesterol 7a-hydroylase, the rate limiting enzyme in bile acid synthesis (41), was not significantly modified by the parasitism, suggesting that increased synthesis was not involved. It is well known that hydrophobic bile acids can promote cxidative stress by disrupting the membrane structure due to their detergent properties (42) and by oxidatively activating Kupffer cells to generate reactive oxygen species (43,44). To investigate if changes in bile acid pool composition could be involved in the development of oxidative stress in infected animals the bile acid profile in bile was studied. Only small changes in the profile of secreted bile acids in infected animals were observed. It has been recently reported in hamster liver microsomes the existence of a non-P450, NADP(+)-dependent 7a-

hydroxycholesterol dehydrogenase which catalyzes the formation of 7-oxocholesterol, which in

turn migh: regulate bile acid metabolism (45). 7oxo,3a,12a dihydroxy, SP-cholanoic acid was the major oxo-bile acid in control hamster bile. This bile acid was increased after infections, the highest proportion found in some infected animals was 2.5% of total bile acids. Thus, increased oxo-bile acid formation seems to be the result rather than the cause of the above-described modification in the redox state of the hepatocyte. A role of parasite metabolism in this change and in the observed slight shift from primary to secondary bile acids cannot be ruled out.

Different studies have shown that intracellular vesicle trafficking plays an important role in delivering both phospholipid and cholesterol to the internal hemileaflet of the hepatocyte canalicular membrane and it is also known that bile acid stimulation of the biliary lipid excretion may occur by enhancement of the transcellular transcytotic pathway (46). Colchicine is a well known microtubule inhibitor that appears to have no effect on basal bile acid secretion or on the secretion of tracer doses of [14C] taurocholate (47,48). In contrast, with increasing doses of bile

1972 Biliary Function in Experimental Dicrocoeliosis Vol. 63, No. 22, 1998

acids there is an inhibition in the secretion of bile acids associated with a parallel decrease in biliary cholesterol and phospholipid output (49). In agreement with previous reports, the results presented here suggest that the transcytotic is of importance for biliary lipid secretion in parasited animals. The mechanism of infection-induced stimulation of colchicine-sensitive bile acid secretion is not known. Moreover, the biological significance of this effect for the parasite physiology is difficult to explain. It must be considered, however, that, in contrast to other hematophagous trematodes such as Fasciola hepatica, Dicrocoelium dendriticum feds on bile, mucine and desquamated epithelial cells (3). Increased secretion of biliary components could thus be proposed to be aimed to increased food sources.

In experiments where exogenous taurocholate infusion was administered, the peak in bile acid secretion in control animals revealed that maximal secretion rate was probably not reached because the basal output (22 nmol/g liver/min) plus the administered bile acid rate (-140 nmol/g liver/min) was not higher than peak in bile acid secretion. By contrast, in infected animals, this value was markedly reduced below the endogenous plus administered bile acid output rate. Thus under these experimental conditions, peak in bile acid secretion can be assumed to reflect maximal secretion rate (SRmax). This has been proposed to be dependent upon the ability of the hepatocyte to restore the continuous release of membrane lipids into bile (50). If transcellular vesicle trafficking is actually stimulated, resulting in an increased continuous output of bile acids, phospholipids, cholesterol and other constituents of the canalicular membrane, such as alkaline phosphatase, the possibility exists that the ability of the hepatocyte to respond to the exogenous taurocholate load-induced demand of repairing lipids transfer from the intracellular stores toward the canalicular membrane might be lowered, and hence SRmax would be also reduced.

Additionally, taurocholate SRmax may be limited by a reduction in the activity of canalicular bile acid transport systems (51). Previous results by Akerboom et al (52) demonstrate that taurocholate canalicular output was closely related to GSSG intracellular content, being decreased upon addition of compounds that increase GSSG levels, such as hydroperoxide or diamide. Thus increase in liver GSSG content may be involved in the reduction in SRmax observed in infected animals.

Bile acid effiux from ductular bile biliary system cannot be ruled out. Cholehepatic shunting of bile acids is consistent with enhanced biliary lipid output (53). However, if this shunt exists, this cannot account for many of the changes in the biliary function that have been observed in infected animals, such as increased bile flow and bile acid output together with absence of both bile acids with high pKa in bile and enhanced bicarbonate secretion.

In summary, although one must be cautious regarding interpretation of the findings because of the presence of the infection and inflammation, these results indicate that, even in the absence of exogenous load of bile acids, stimulation of colchicine-sensitive mechanisms may substantially contribute to bile acid secretion. This probably accounts for Dicrocoelium dendriticum-induced enhancement in the biliary output of bile acids, cholesterol, phospholipids and canalicular membrane proteins, which play a nutritional role in the physiology of the parasite living downstream. Metabolic changes induced in the liver by the presence of the parasites, also contribute to canalicular bile formation by increasing glutathione output. Moreover, dilution of canalicular bile with nutrient-poor ductular secretion is reduced in infected animals. Whether dicrocoeliosis-induced changes in the biliary function of the host has been selected during evolution because they play a role in the parasite physiology or they are mere epiphenomena of the infective process is an interesting question that deserve further investigation.

Vol. 63, No. 22, 1998 Biliary Function in Experimental Dicroeoeliosis 1973

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