Hepatic disposition of organic anions in rats infested with Fasciola hepatica

EXPERIMENTALPARASITOLOGY

Hepatic

73,39wO2(1991)

Disposition

P. GONZALEZ, M.J. Department

of Physiology,

of Organic Anions Fasciola hepatica TUAON, P. LOPEZ,N.

in Rats Infested

with

DIEZ,* AND J. GONZALEZ

Pharmacology and Toxicology, and *Department University of Ledn, Spain

of Animal Pathology,

GONZALEZ, P., TUCSON, M. J., LOPEZ, P., DIEZ, N., AND GONZALEZ, J., 1991. Hepatic disposition of organic anions in rats infested with Fascio[a hepatica. Experimental Parasitology, 73, 396402. This study was undertaken to investigate the effects of subclinical fascioliasis at various stages of its development (3, 6, and 11 weeks after infestation with an oral dose of 20 metacercariae of Fasciola hepatica) on the hepatobiliary transport of organic anions in rats. Basal bile acid secretion was not significantly modified in infested rats as compared to the controls but there was a significantly increased bile flow in the Il-week group. In all infested rats, maximal biliary excretion and cumulative excretion of bromosulphthalein was significantly reduced, although there was a tendence to recovery in the 1 l-week infested group. Decreased biliary secretion was accompanied by increased plasma and liver concentrations of bromosulphthalein. Glutathione S-transferase activity was significantly reduced by Weeks 3 and 6 postinfestation. Liver glutathione concentration was significantly reduced in all infested rats. Maximal excretion of dibromosulphthalein, a compound not biotransformed in the liver, fell by Week 6 postinfestation and recovered a normal value thereafter. These results indicate that effects of experimentally induced fascioliasis on hepatic disposition of organic anions depend upon the stage of its development and that are 0 1991 Academic Press, Inc. a consequence of both metabolism and transport related factors. INDEX DESCRIPTORS AND ABBREVIATIONS: Fasciola hepatica; Trematode, parasitic; Rat; Metabolism, hepatic; Bile; Bromosulfthalein (BSP); Dibromosulfthalein (DBSP); Glutamic piruvic transaminase (GPT); Glutamic oxalacetic transaminase (GOT).

cantly reduced both in rats (Galtier et al. 1987) and lambs (Galtier et al. 1986) but contradictory reports have been given with respect to UDP-glucuronyl transferase activities (Facino et al. 1985; Galtier et al. 1987). In the only study about the effect of subclinical fascioliasis on biliary excretion of drugs, it has been reported that the excretion of rafoxanide is not significantly altered in rats, while there is a significant decrease in that of tetracycline (Galtier et al. 1985b). The present study was undertaken to evaluate the possible sites of action of experimental fascioliasis, at various stages of its development, on the hepatic transport of organic anions. The conjugation and canalicular excretion of BSP, an anionic dye that has been extensively used as a model for studying hepatobiliary function, were investigated in rats. Dye levels in plasma,

In spite of the frequent occurrence of liver parasitism by Fusciola hepatica in both humans and breeding animals, there has been a lack of information about the effects of this infection on the hepatic disposition of drugs and other xenobiotics. Different studies have demonstrated, however, alterations of the hepatic drug biotransformation system both in rats and in cattle. Thus, subclinical fascioliasis induced experimentally has been shown to significantly decrease liver microsomal cytochrome P450-dependent monooxygenases (Galtier et al. 1986) and antipyrine clearance (Tufenkji et al. 1988) in sheep. In rats, some monooxygenase activities are inhibited (Galtier et al. 1983), while others are not affected (Galtier et al. 1985a). Glutathione S-transferase activities are signifi396 0014-4894/91 $3.00 Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.

FASCIOLIASIS

AND DISPOSITION OF ORGANIC ANIONS

liver, and bile, as well as liver glutathione concentration and glutathione S-transferase activities were determined to define possible changes in the different steps of the hepatic transport of this organic anion. The effect on DBSP excretion was also determined to further assess alterations of nonmetabolic components of the hepatic excretory mechanism, as DBSP competes with BSP for various steps in hepatic transport but is not metabolized (Vonk et al. 1979). MATERIALSAND

METHODS

Chemical. Bromosulphthalein, 5,5dithiobis(2nitrobenzoic acid), reduced glutathione, glutathione reductase, 3-a-hydroxysteroid dehydrogenase, nicotinamide adenine dinucleotide, and nicotinamide adenine dinucleotide phosphate were purchased from Sigma Chemical Co. (St. Louis, MO). Dibromosulphthalein was obtained from SERB (Paris, France). Pentobarbital sodium was purchased from Claudio Barcia (Madrid, Spain). All other reagens were of the highest quality available. Animal treatment. Male Wistar rats (160-220 g body wt) were housed in cases in a room at 22°C and with a 12-hr dark/light cycle. Each infested rat received, by gastric tubing, 20 metacercariae of F. heparica suspended in a 1% Tween aqueous solution. The metacercariae used to infect the rats were recovered from Lymnaea truncatula and maintained in artificial culture. Parallel studies were carried out on uninfected control animals. Experiments were carried at 3, 6, and 11 weeks postinfestation. Experimental procedures. Ail surgery was cat+ed out under pentobarbital anaesthesia (50 mg/kg body wt ip). Rectal temperature was monitored via a thermistor probe and maintained at 37°C. After catheterization of the right jugular vein and right carotid artery, a midline abdominal incision was made and the bile duct was cannulated with polyethylene tubing (PE-50). After collecting two baseline samples of bile, both control and infested animals received either BSP or DBSP. The dyes (dissolved in 0.145 M NaCl) were administered as an iv injection of 2.15 pmol/lOO g body wt followed by a 60-min infusion at 215 nmol/lOO g . min body wt. Bile samples were collected under ice at lo-min intervals. At the end of the experiments a blood sample was obtained from the carotid artery catheter, the animals were killed by exanguination, and the livers were excised and weighed. A specimen of each liver was fixed in 4% buffered formalin for histological examination. Analytical procedures. Bile flow was measured gravimetrically assuming a bile density of 1.0 g/ml.

397

The concentration of BSP and DBSP in plasma and bile was determined spectrophotometrically at 580 nm after appropriate dilution with 0.05 N NaOH. Liver BSP and DBSP concentrations were measured by the method of Whelan and Combes (1971). Free and conjugated BSP were separated by paper chromatography using as solvent system butan-1-ohacetic acid/ethanol/ water (120/l/120/40). The spots were cut, eluted with water, and read at 580 nm after alkalinization. Bile acid concentration in bile was determined by the method of Paungartner et al. (1971). Determination of total glutathione content of liver homogenates prepared in cold 5% (w/v) thrichloroacetic acid in 0.01 N HCI was measured as described by Tietze (1969) with the modification of Griffith (1980). Glutathione S-transferase activity was determined using BSP as substrate (Goldstein and Combes, 1966). Protein concentration in the liver was determined by the method of Lowry et al. (1951). The activities of plasma glutamic transaminase (GOT) and plasma glutamic piruvie transaminase (GPT) were estimated by commercially available kits (Boehringer-Mannheim, Germany). Statistical methods. Means and SEMs were calculated for all data. Significant differences were determined using the Mann-Whitney U test. Values lower than 0.05 were considered to be significant. RESULTS

The presence of subclinical fascioliasis was confirmed by the recovery of fluke eggs in faeces of animals from 6 weeks after infestation and by plasma enzyme data. Both GOT and GPT activities were significantly increased by Weeks 3-6 postinfestation (Table I). The ratio of liver to body weight was significantly increased only in the 6-week infested group (4.50 ? 0.6 g/100 g vs 3.37 k 0.3 g/100 g; P < 0.05). Body weight increase did not significantly differ between control and infested animals for the 11-week experimental period (data not shown). The mean rate of bile flow in the period prior to the administration of organic anions is shown in Table I. Bile flow was not significantly modified by Weeks 34 postinfestation but there was a significant increase by Week 11. No significant changes in bile acid secretion were found in any group of infested animals as compared to the controls (Table I).

398

GONZALEZ ET AL.

TABLE I Changes in Plasma Enzyme Activities and Basal Bile Flow and Bile Acid Secretion during the Course of Experimental Fascioliasis in rats Weeks postinfestation Control GOT (U/l) GPT (U/l) Bile flow (p.l/lOO g min) Bile acid (nmol/lOO g . min)

107 ? 23 k 5.7 k 238 2

IO 2 0.4 21

3 324 2 75 2 5.7 2 205 k

6 18* 13* 0.5 31

11

91.52 101* 91 k 28* 5.4 -+ 0.5 228 t 25

197 2 25 k 6.5 2 225 k

39* 6 0.3* 19

Note. Values are means 2 SEM from five animals. Plasma data were obtained at the end of experiments. Biliary data correspond to the 0- to 20-min basal period. * P < 0.05 significantly different from control value

Following BSP administration, maximal excretion of the dye was reduced by 3 (- 40%), 6 (- 54%), and 1I (- 24%) weeks postinfestation (Fig. 1). Cumulative biliary

,o-pJ-y-y

20

40

60

Time(min) FIG. 1. Effect of experimental fascioliasis on bile concentration and excretion of BSP and on bile flow. Values are means + SEM from five animals and correspond to the 60-min period following BSP administration. *P < 0.05 significantly different from control value.

excretion was also significantly reduced by Weeks 3 (-31%), 6 (-52%), and 11 ( - 22%) (Fig. 3). The depression in BSP excretion was due to an inability to concen-

r

L-l ‘d

3 week

n

6 week

i% II week

20

40 Time (mid

60

FIG. 2. Effect of experimental fascioliasis on bile concentration and excretion of DBSP and on bile flow. Values are means + SEM from five animals and correspond to the 60-min period following DBSP administration. *P < 0.05 significantly different from control value.

FASCIOLIASIS

399

AND DISPOSITION OF ORGANIC ANIONS

trate BSP in bile (Fig. 1). Bile flow decreased in parallel to the changes detected in the excretion of the organic anion (Fig. 1). The decrease in BSP excretion could be accounted for a lowered excretion of the conjugated dye, with no significant change in that of unconjugated BSP (Table II). In all groups of infested rats plasma and liver BSP concentrations were significantly increased as compared to control animals (Table III). Glutathione S-transferase activity was significantly lowered by 3 and 6 weeks postinfestation (Table II) while glutathione content in the liver remained significantly lowered in the groups of infested rats (Table II). When animals were administered DBSP there was no significant change in the maximal biliary excretion (Fig. 2) and cumulative excretion (Fig. 3) of the organic anion by Weeks 3 and 11, but maximal biliary excretion was significantly reduced by Week 6 (Fig. 2). Plasma DBSP concentration did not significantly differ in any of the groups of rats. Liver concentration of the dye was significantly higher by Week 6 (Table III). DISCUSSION

Hepatic transport function for BSP can be affected at various levels, including membrane transport, intracellular metabo-

lism, and biliary excretion. Changes in one or more of these processes could explain the reduced biliary excretion of BSP in rats infested by F. hepatica metacercariae. It is well known that the excretion of organic anions may be modified by changes in bile flow and/or bile acid secretion. It has been reported that biliary excretion of BSP is increased following infusion of bile acids (Gregus and Fischer 1979) or reduced after bile acid depletion by cholestyramine treatment (Gregus et al. 1980). However, in our experiments there was no significant change in bile acid secretion regardless of the stage of parasitic infestation. Bile flow was significantly modified by Week 11, but data from different authors indicate that both bile-acid-dependent or independent bile flow changes do not apparently alter the maximal biliary excretion of conjugated BSP (Barnhart and Combes 1976; Fischer and Varga 1984). The increased bile flow without changes in bile acid secretion is an interesting phenomenon that has also been reported in calves after 10 weeks of F. hepatica infestation (Symonds et al. 1983). Our data indicate that migration of flukes in the common bile duct apparently stimulates the bile-acid-independent fraction of bile flow. Although the exact mechanism responsible for this effect requires further investigation,

TABLE II Changes in Liver Glutathione S-Transferase Activity and Free and Conjugated BSP in Bile during the Course of Experimental Fascioliasis in Rats Weeks postinfestation

Glutathione (pmoUg liver) Glutathione S-transferase (nmol/g liver . min) Conjugated BSP (nmol/lOO g . min) Unconjugated BSP (nmol/lOO g . min)

Control

3

6

11

2.69 + 0.26

2.16 2 0.18*

1.65 2 0.27*

2.00 -c 0.32*

0.63 k 0.04

0.43 k 0.06*

0.50 2 0.06*

0.58 -c 0.04

102 + 14

58 2 7*

44 k 8*

77 -c 13

12 rt 2

11 -r- 1

9-r-l

10 -c 2

Note. Values are means f SEM from five animals. Liver data were obtained at the end of experiments. Biliary data correspond to the 20- to 40-min period after beginning of infusion. *P < 0.05 significantly different from control value.

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GONZALEZ ET AL.

Changes in Plasma and Liver BSP

and

TABLE III DBSP during the Course of Experimental Fascioliasis in Rats Weeks postinfestation

Control Plasma BSP (mmoVliter) Liver BSP (pmol/g liver) Plasma DBSP (mmohliter) Liver DBSP (pmohg liver)

0.18 2 0.26 5 0.45 t 0.13 ”

0.02 0.02 0.07 0.01

6

3 0.25 f 0.44 2 0.48 2 0.13 2

0.04* 0.10* 0.06 0.03

0.25 0.45 0.43 0.16

2 2 + 5

11 0.03* 0.09* 0.06 O.Ol*

0.27 k 0.35 2 0.54 + 0.12 2

0.04* 0.09* 0.06 0.01

Note. Values are means 2 SEM from five animals and were obtained at the end of experiments. *P < 0.05 significantly different from control value.

it could be related to the increase in the number of epithelial cells and the hyperplastic response of the biliary epithelium (Foster, 1981). A similar increased choleresis has been also found in the latter stages of Eimeria stiedaii infection in rabbits (Esteller et al. 1990). Different studies have demonstrated that changes in BSP transport could be mediated by changes in its rate of metabolism. Rates of conjugation can be modulated by either enzyme activity or cosubstrate concentration. It is known that BSP excretion -

FIG. 3. Effect of experimental fascioliasis on cumulative excretion of BSP and DBSP. Values are means + SEM from five animals. 'P < 0.05 significantly different from control value.

is decreased when glutathione S-transferase is inhibited by tienilic acid (Fehring and Ahokas 1989), hypolipidemic drugs (Foliot et al. 1984), perhexiline maleate (Mariscal et al. 1988), and clotrimazole (Tunon et al. 1989). An impairment in BSP excretion following glutathione depletion has also been reported (Aza et al. 1986). Depletion of cytosolic glutathione induced by experimental fascioliasis has been previously found in lambs and explained on the basis of oxidative cell injury as a consequence of tissue destruction by the young flukes (Galtier et al. 1986). This parasitosis has also been found to cause a reduction in glutathione S-transferase activity both in lambs (Galtier et al. 1986) and in rats (Galtier et al. 1987). Data in the literature are confirmed by our work. In the present study both glutathione S-transferase activity and glutathione concentration were significantly reduced by 3-6 weeks following infestation and glutathione concentration remained depressed even in the 11-week period. Thus, a reduced metabolic rate could account for the impaired biliary excretion of BSP in the acute stage of the disease and for its still uncomplete recovery in the chronic stage. This explanation coincides with previous results demonstrating a parallel fall in plasma clearance and elimination rate constant of BSP by Weeks 8-16 postinfestation in lambs (Tufenkji et al. 1987). The parallel decrease in bile flow and BSP excretion in infested animals could be

FASCIOLIASIS

AND

DISPOSITION

OF ORGANIC

ANIONS

401

explained on the basis of the reduced biliIn summary, our data indicate that there ary excretion of the organic anion. It is is a complex interaction between experiknown that conjugated BSP has a choler- mental fascioliasis and the hepatobiliary etic effect on rats (Fischer er al. 1978) and transport of organic anions and confirms at secretion of the conjugated dye is markedly the level of the biliary secretory step a disdepressed in infested animals. Additionturbed capacity for handling of drugs and ally, the reduced conjugation of BSP would xenobiotics. The effects depend upon the increase hepatic concentration of unconju- stage of development and seem to be a congated BSP, which has been shown to inhibit sequence of both metabolism and transport related factors. The importance of this phemithochondrial respiration and the activity nomenon for a correct dosage of antihelmof membrane ATPases (Aza et al. 1986). In spite of the apparent relationship be- intic agents must be considered. tween decreased conjugation and biliary excretion of BSP, the possibility that REFERENCES changes in transport processes could contribute to the impairment of the organic an- AZA, M. J., GONZALEZ, J., AND ESTELLER,A. 1986. Effect of diethyl maleate pretreatment on biliary exion disposition cannot be ruled out. Altercretion and choleretic action of sulfobromophthalein ations of transport mechanisms per se are in rats. Archives Internationales de Pharmacodyapparently responsible for a reduced excrenamie et de Therapie 281, 321-336. tion of BSP in different situations such as BARNHART, J. L., AND COMBES,B. 1976. Biliary excretion of dye in dogs infused with BSP or its gluaging (Kanai er al. 1988) or administration tathione conjugate. American Journal of Physiology of a variety of drugs (Hoyt and Larson 231, 399-407. 1989). ESTELLER, A., TORRES, M. D., GOMEZ-BAUTISTA, To assessthe actions of experimental fasM., MARINO, E. L., FERNANDEZ-LASTRA,C., AND cioliasis on nonmetabolic components of JIMENEZ, R. 1990. Pharmacokinetics, hepatic organic anion excretion, biliary excretion biotransformation and biliary and urinary excretion of bromosulphthalein (BSP) in an experimental liver of DBSP was investigated. Although this disease mimicking biliary cirrhosis. European Jourdye shares the same transport system with 15, nal of Drugs Metabolism and Pharmacokinetics BSP, it is rapidly excreted into bile without 7-14. requiring further metabolism (Vonk et al. FACINO, R. M., CARINI, M., AND GENCHI, C. 1985. 1979). Our data indicate that at least in the Decrease in hepatic microsomal UDP-glucuronosyltransferase activity in rats and cattle with fascioliaperiod 6 weeks after infestation, maximal sis: Impaired in vitro glucuronidation of oxyclozaexcretion of DBSP was significantly renide. Toxicology Letters 26, 65-71. duced. This stage corresponds to the wanFEHRING, S., AND AHOKAS, J. T. 1989. Effect of the dering of young flukes through the hepatic glutathione S-transferase inhibitor, tienilic acid, on parenchyma before their passage to the bile biliary excretion of sulfobromophthalein. ChemicalBiological Interactions 69, 23-32. ducts and coincides with the maximal increase in plasma GOT and GPT activities. FISCHER,E., GREGIJS,Z., AND GOGL, A. 1978. Bile flow and biliary excretion rate of some organic anAlthough the exact mechanism involved in ions in phenobarbital-pretreated rats. Digestion 17, the impairment of DBSP excretion is difti21l-220. cult to elucidate, most probably there is an FISCHER,E., AND VARGA, F. 1984. Effect of tauroalteration of transport processes across celcholate pretreatment on the excretion of exogenous organic anions in rats. Archives Znternationales de lular membranes. A similar effect has been Pharmacodynamie et de Therapie 267, 1287-1298. previously reported for the hepatic disposition of tetracycline in the rat and attributed FOLIOT, A., TOUCHARD, D., AND CELIER, C. 1984. Impairment of glutathione S-transferase activity as a to a decreased uptake and decreased efficause of reduced biliary sulfobromophthalein excreciency of the secretory process as a consetion in clofibrato-treated rats. Biochemical Pharmaquence of alterations of the structural integcology 33, 2829-2834. FOSTER,J. R. 1981. A study of the initiation of biliary rity of the liver (Galtier et al. 1985b).

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1991; accepted with revision

1