Camp. Biochem. Physiol. Vol. 103C, No. I, pp. 53-56, 1992 Printed in Great Britain

0306-4492/92 $5.00 + 0.00 Q 1992 Pergamon Press Ltd

ALTERATIONS INDUCED BY FASCIOLIASIS AND CIRRHOSIS ON THE BILIARY EXCRETION OF

CEFMETAZOLE IN WISTAR RATS

PILAR LOPEZ, LUIS A. GARCIA-PARDO, JAVIER GONZALEZ-GALLEGO, PAQUITA GONZALEZ and MARIA J. TUGON*

Department of Physiology, Pharmacology and Toxicology, University of Le6n, Spain (Tel.: 3487-291-261; Fax: 3487-291-194)

(Received 18 February 1992; accepted for publication 25 March 1992)

Abstract-l. Alterations induced by fascioliasis and cirrhosis on the biliary excretion of cefmetazole have been studied in Wistar rats.

2. Both infestation with Fasciolu heputica and experimental cirrhosis originated a significant decrease in the biliary excretion and in bile flow increase induced by the drug.

3. Administration of the p-lactam antibiotic induced a lower degree of uncoupling of biliary lipid secretion in the cirrhotic and fasciolotic animals. but the effect was evident in all experimental groups.

INTRODUCTION

Bile flow and composition in mammals and other vertebrates is modified by a number of xenobiotics. These effects depend on the magnitude of biliary excretion and the interactions with bile formation mechanisms and may present profound variations under the presence of different hepatobiliary diseases.

Information on the effects of the parasitism by Fasciola hepatica on the hepatobiliary transport of drugs and other xenobiotics is scanty in spite of the high incidence of this infestation both in humans and domestic animals. Different studies suggest alter- ations of the hepatic biotransformation systems in rats and sheep (Galtier et al., 1983, 1985b, 1986, 1987; Tufenkji et al., 1988). Rafoxanide biliary excretion is not modified in the rat (Galtier et al., 1985a) while that of tetracyclin and bromosulphthalein is markedly reduced (Galtier et al., 1985a; Gonzblez et al., 1991).

Hepatic cirrhosis courses with reduction of hepatic clearance of many endo- and xenobiotics both in humans (Pessayre et aI., 1978; Huet and Villeneuve, 1983) and laboratory animals (L6pez et al., 1991; Reichen et al., 1987). Mechanisms responsible for these alterations are unknown at present. The fact that clearance of compounds with a low hepatic extraction is similarly affected than those with a high hepatic extraction led to the operational model of the “intact cell hypothesis” (Wood et d., 1979). The “sick cell hypothesis”, however, proposes an abnormal function of individual hepatocytes (Huet y Villeneuve, 1983; Villeneuve et al., 1978).

Data on the influence of hepatobiliary diseases in the disposition of antibiotics presenting a biliary excretion are specially rare and in some cases conflict- ing. Cefmetazole is a third generation cephalosporin

*To whom all correspondence should be addressed at Departamento de Fisiologia, Farmacologia y Toxico- lo&a, Facultad de Veterinaria, 24071 Le6n, Spain (Tel.: 34 87 291260; Fax: 34 87 291194).

eliminated in active form both by renal and hepatic pathways, that present no hepatic biotransformation (Gondlez et al., 1989). Percentage of biliary excre- tion varies from different species of mammals, being low in dogs or monkeys but markedly high in the rat (Gonzblez et al., 1989; Shindo et al., 1982). The purpose of this study was to examine the possible alterations caused by the two above mentioned liver diseases, fascioliasis and cirrhosis, in the biliary excretion of cefmetazole in the Wistar rat.

MATERIALS AND METHODS

Animal treatment

Male Wistar rats (Charles River, Barcelona, Spain) were housed in a room maintained on a 12 hr light/l2 hr dark cycle and at 22-23°C. The rats were allowed free access to pelleted food and water throughout.

Rats received 20 metacercariae of Fuxiolu hepatica sus- pended in a 1% Tween aqueous solution, by gastric tubing, to induce fascioliasis. The metacercariae used to infest the rats were recovered from Lymnaea truncutula and maintained in artificial culture. Parallel studies were carried out on uninfected control animals. Experiments were carried at 6 weeks postinfestation.

Cirrhosis was induced by chronic exposure to drinking water containing phenobarbital and by gassing with Ccl, as described by McLean et al. (1969). Treatment was carried out for 11 consecutive weeks and was ceased 14 days before the study. Control animals were kept under the same conditions without any treatment.

Experimental procedures

All surgery was carried out under pentobarbital anaesthe- sia (50 mg/kg body wt i.p.). Rectal temperature was moni- tored via a thermistor probe and maintained at 37°C. After catheterization of the right jugular vein, a midline abdomi- nal incision was performed and the bile duct was cannulated with polyethylene tubing (PE-50).

After collecting two basline samples of bile, control, infested and cirrhotic animals received an iv. injection of 2OOpmol/kg body wt of cefmetazole. Nine additional IO-min samples were collected.

53

54 F?LAR Lcmz et al.

s

j:: ;;

90 I10

TIME (mio)

Fig. 1. Effect of fascioliasis and cirrhosis on the biliary excretion of cefmetazole (CMZ). Values are means & SEM from 5-7 animals. *P < 0.05 Significantly different from

control values.

A specimen of the liver from each cirrhotic animals was fixed in 4% buffered formalin for histological examination.

Analytical procedures

Bile flow was measured gravimetrically assuming a bile density of 1 .O g/ml. The concentration of cefmetazole in bile was determined by a high-performance liquid chromato- graphic method with a reversed-phase technique. A Spectra- Physics (San Jose, CA) chromatograph system with a UV/ visible detector (model SpectraChrom 200) set at 254 nm, a peak integrator (model ChromJet) and a loop injector of fixed volume (20 ~1) was used. Conditions were as follows: column, ~Bondapak C,,; mobile phase, methanol-phosphate buffer (0.007 M, pH 7.4, 18/82 (vol/vol)); and flow rate, 1.0 ml/min. The detection limit was 0.1 pg of cefmetazole per ml. Bile acids in bile were determined with 3cc-hydroxy- steroid dehydrogenase by the method of Talalay (1960). Biliary cholesterol and phospholipid concentrations were measured by enzymatic methods (Bolton ef al., 1980; Gurantz et al., 1981).

Statistical procedures

Values are presented as means k SE of the mean. Significance of the differences between means was evaluated by the non-parametric Mann-Whitney U test. P values less than 0.05 were considered to be significant.

RESULTS

Figure 1 presents the biliary excretion of cefmeta- zole in the different experiments. Maximal antibiotic

50 _I

3

: 40-

s

% 30- F

3 ” zo-

t:

5 IO-

. Fascioliasis

0-l : , , , . 30 50 10 90 110

TIME(min)

Fig. 2. Effect of fascioliasis and cirrhosis on the cumulative biliary excretion of cefmetazole (CMZ). Values are means f SEM from 5-7 animals. *P < 0.05 Significantly different

from control values.

‘i OT , I , ( , I

30 50 70 90 110 TIME(min)

Fig. 3. Effect of fasciohasis and cirrhosis on cefmetazole- induced choleresis. Values are means + SEM from 5-7 animals. *P < 0.05 Significantly different from control

values.

biliary excretion (Tm) in all the groups was reached 20 min after the injections. Values were significantly reduced both in parasited (-67%) and cirrhotic (- 38%) animals. Cumulative biliary excretion of cefmetazole was 41.1 pmol in control animals, that amounts to 54.5% of the administered dose (76 Bmol). In animals infestated with Fusciola hepatica cumu- lative excretion reached only a 24.5% of the dose. In cirrhotic animals a 40.7% of the administered dose was found in bile (Fig. 2).

Administration of cefmetazole produced a marked choleresis in control rats (Fig. 3). A significant choleretic effect was also found in animals with both diseases, although maximal bile flow values were lower than those found in the controls.

Figure 4 shows changes in the biliary secretion of bile acids, cholesterol and phospholipids during the period of maximal cefmetazole excretion as compared to the basal period. In control animals cholesterol and phospholipid secretion were markedly reduced following administration of the antibiotic, without changes in bile acid secretion. Similar results were obtained both in parasited and cirrhotic animals, although phospholipid secretion in both groups and cholesterol secretion in cirrhotic animals were reduced to a lower extent than in the controls.

100

I 0 0 Control

B : El . Fascioliasis

l Cirrhosis

. l

I w

a *

0

0

“I

Bilcacid Cholcstcrol Phospholipid

Fig. 4. Effect of cefmetazole administration on the biliary secretion of bile acids, cholesterol and phospholipids in the different experimental groups. Data correspond to mean values (5-7 animals) for 30-40 min of experiments and are expressed as percentage of the basal period (O-20min) values. *P -C 0.05 Significantly different from control values.

Fascioliasis, cirrhosis and cefmetazole 55

DISCUSSION

Our data indicate that the biliary excretion of

cefmetazole in the Wistar rat is reduced in the liver diseases, fascioliasis and cirrhosis. The exact mech- anisms responsible for these effects are difficult to elucidate.

Forty days after experimental infestation in the rat, young fasciolas migrate through the hepatic parenquima causing an acute inflammation of the hepatic tissue due both to mechanical factors and to the release of toxic metabolites (Galtier et al., 1983). These effects could explain the alterations in the hepatobiliary transport of the antibiotic and the significantly reduced excretion in infestated animals.

The exact mechanisms responsible for these effects are difficult to elucidate. In the case of cirrhosis, some authors postulate that the impairment in the excretory function of the liver could be the result of a reduced number of hepatocytes with normal func- tion and of hemodynamic alterations (Meyer et al.,

1990; Reichen et al., 1987). Other studies propose an abnormal function of individual hepatocytes (Pessayre et al., 1978; Huet and Villeneuve, 1983). Additional studies using isolated hepatocytes would be required to further establish the factors respon- sible for the reduced hepatobiliary transfer of cefmetazole.

Following cefmetazole injection a marked choleretic effect in the control group was found. This effect has been previously described and appears to be a conse- quence of the stimulation of the bile acid-independent fraction of bile flow, with no change in the secretion of bile acids (Gonzhlez et al., 1989). The increase in bile flow amounted to lower values in parasited and cirrhotic animals, an effect probably due to the significantly reduced excretion of the drug.

Our results indicate that endovenous administration of cefmetazole impairs the secretion of phospholipid and cholesterol with no changes in bile acid secretion. This uncoupled secretion of biliary lipids in the rat has been previously reported for this antibiotic (Cava et al., 1991) as well as for other xenobiotics such as bilirubin, ampicillin or cefoperazone (Apstein, 1984; Apstein and Russo, 1985; Pattinson et al., 1987). The effects in the case of cefmetazole are apparently not due to canalicular but rather to presecretory events (Cava et al., 1991). Although phospholipid and cholesterol secretions were also reduced in fascioliasis and cirrhosis, the inhibitory phenomenon was less conspicuous than in the case of control animals. This could be based on the impaired hepatic handling of the drug and, as a consequence to a lesser interference between the hepatobiliary transport of cefmetazole and the secretion of biliary lipids.

In summary, data presented in this study indicate that both fascioliasis and cirrhosis impair the hepatic handling of cefmetazole in Wistar rats. The decreased microsomal oxygenase activity reported by other authors in animals infestated by Fasciola hepatica

(Galtier et al., 1985a) together with the results presented here, are important data to consider when adjusting therapeutical doses for drugs used in the course of the infestation, in order to avoid potential toxicity. Similarly, prolongation of the half-life and increases in the plasmatic concentration of

drugs in cirrhotic animal may also cause systemic toxicity.

Acknowledgemenr-This research was supported in part by a grant from the Plan National de Investigaciones Ganaderas, Spain.

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