Effects of mebendazole on the absorption of low molecular weight nutrients by Ascaris suum

International Journal for Parasitology, 1973, VoL 3, pp. 401-407. Pergamon Press. Printed in Great Britain

EFFECTS OF MEBENDAZOLE ON THE ABSORPTION OF LOW MOLECULAR WEIGHT NUTRIENTS BY ASCARIS SUUM* H. V A N D E N B O S S C H E and S. D E N O L L I N Department of Comparative Biochemistry, Janssen Pharmaceutica, Research Laboratories, B-2340 Beerse, Belgium (Received 13 September 1972; amended 23 October 1972)

Abstract

VANDEN BOSSCHEH. and DE NOLLINS. 1973. Effects of mebendazole on the absorption of low molecular weight nutrients by Ascaris suum. International Journal for Parasitology 3: 401-407. The effect of the anthelmintic drug, mebendazole, on the uptake and/or transport of glucose, fructose, 3-O-methylglucose, glycine, proline, methionine and palmitic acid was studied on in vitro incubated Ascaris suum. The experiments presented indicate that mebendazole inhibits the uptake and/or transport of glucose by A. suum. This inhibition is followed by a marked decrease in the glycogen content of the ascaris muscle. The addition of glucose to the incubation medium significantly enhanced the rate of uptake and/or transport of 3-O-methylglucose, glycine, methionine, proline and palmitic acid indicating that the absorption mechanisms depend on energy. Therefore, the inhibitory effect of mebendazole on the glucose uptake also results in a decreased uptake of 3-O-methylglucose and of the amino acids and fatty acid studied. The fructose uptake was not affected by the addition of glucose. Although mebendazole decreased the uptake of the hexoses and of the amino acids whether or not glucose was added, the uptake of palmitic acid was not affected when glucose was omitted from the medium. Mebendazole failed to exhibit an effect on the uptake, transport and/or utilization of glucose in rat. INDEX KEY WORDS: Ascaris suum; mebendazole; glucose; fructose; 3-O-methylglucose; glycine, methionine; proline; palmitic acid; glycogen; muscle; intestine; pseudocoelomic fluid; reproductive system; absorption mechanisms.

INTRODUCTION THE ANTHELMINTICdrug, mebendazole/f inhibits the glucose uptake by adult nematodes at concentrations which do not affect the motility o f these parasites. As a result o f the inability to use exogenous glucose, the endogenous glycogen reserves in the muscle tissue are utilized. This leads to a decreased generation o f energy-rich phosphate bonds (Van den Bossche, 1972). Experiments described in this report were designed to answer a n u m b e r o f questions a b o u t the specificity o f the mebendazole induced inhibition o f the glucose uptake. It is necessary to determine whether this anthelmintic also interferes with the glucose uptake by the host. It also seemed essential to k n o w whether the effect o f mebendazole on the parasites is limited to inhibition o f the glucose absorption. Therefore, the inhibitory effect o f this benzimidazole on the uptake of fructose, 3-O-methylglucose, glycine, proline, methionine and palmitic acid were studied. Since the Ascaris intestine is the principal route for the absorption o f nutrients (von Brand, 1972) it can be expected that residual a m o u n t s o f * This investigation was supported by Grant No. D 1/4-1644 from the 'Instituut tot Aanmoediging van het Wetenschappelijk Onderzock in Nijverheid en Landbouw (IWONL)'. I" Mebendazole is the generic name for methyl-5(6)-benzoyl-2-benzimidazole carbamate. 401

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mebendazole should be found in the intestinal cells. The uptake and distribution of mebendazole in the different organs of Ascaris was measured. The effect of mebendazole on the glycogen content of muscle- and intestinal-cells was also studied. MATERIALS

AND

METHODS

A. Preparation o f Ascaris for absorption studies Adult female ,4scaris suum were collected at a local slaughterhouse and transported in a medium which had the following composition: 0.14 M NaCI; 0.0027 M KC1; 0.0018 M CaCI2 and 0.0004 M MgSO4. The temperature was maintained between 30 and 35°C. The worms were washed and incubated in a salt medium, modified from that of Bueding et aL (1961) which had the following composition: 0-12 M NaCI; 0.005 KC1; 0.001 M CaCI2; 0.001 M MgCI2; 0"005 M potassium phosphate buffer (pH = 7.4); 0.045 M NaHCO3. Streptomycin (0"1 g) and penicillin (4 × 103 i.u.) were added to 1 1. of medium. This medium will be referred to as medium A. Medium A. + 0.016 ~l glucose becomes medium B. Incubation was carried out for 20 h at 37°C in an atmosphere of 95 % N2:5 % CO2. B. Uptake studies of low molecular weight nutrients by A. suum The uptake of proline, methionine, glycine, palmitic acid, 3-O-methylglucose and fructose was studied in the absence or presence of D-glucose. A. suum were incubated in 100 ml of medium A or B to which either 0.877 m.~ L-proline plus 1.25 mCi L-proline-14C (U); 0.670 ram L-methionine plus 250 nCi L-methinnine-3'S; 1.33 mM glycine plus 625 nCi glycine-t4C (U); 32.3 pM palmitic acid plus 2.78 pCi palmitic acid-t4C (U); 0.555 mM D-fructose plus 2"5 ~,Ci D-fructose-l'*C (L0 or 1 mM 3-O-methyl glucose plus 10 pCi 3-O-methylx'~C-D-glUCOSewas added. Palmitic acid was present in complex with sodiumglycocholate (2 n'~). The preparation of the bile salt solution was according to Hofmann (1963). Mebendazole was dissolved in dimethylsulfoxide (DMSO) and added to the medium in which one female worm was incubated at 37°C for 24 h in an atmosphere of 95 % N2: 5 Yo CO2. (Final DMSO concentration = 0.1 per cent). After incubation the worms were washed thoroughly and homogenized (Ultra-Turrax) in water to a final volume of 30 ml. One millilitre of the homogenate was placed in a scintillation counting vial and digested according to Mahin & Lofberg (1966) with 0-2 ml HC10,, (70~o) and 0.4 ml H202 (30%) at 70°C. Fifteen millilitres of scintillator solution (Insta-Gel, Packard) was added and the radioactivity determined using a Packard Tri-Carb Liquid Scintillation Spectrometer. Correction for quenching was applied by internal standardization. The incubation medium was centrifuged at 1000 g for 10 rain to collect the eggs. The glucose content of the supernatant was determined by the GOD-Perid method (Boehringer-Mannheim Cat. no.: 15754). C. Distribution of mebendazole in the different organs of A. suum Incubation circumstances were as described above. 14C-labelled mebendazole (specific activity: 0.9 mCi/mM) was dissolved in DMSO (final drug concentration: 0.5 pg/mi of incubation mixture). After 24 h of incubation the worms were dissected and the different organs were collected and homogenized in an isopropanol-H20-HCl mixture (70:30:1). One millilitre of the collected pseudocoelomic fluid and of the different homogenates were placed in a scintillation vial and further treated as described for the experiments with low molecular weight nutrients. D. Uptake of D-glucose by rings of rat small intestine The experiments were performed by incubation in vitro of rings of everted rat small intestine (jejunum) as described by Crane & Mandelstam (1960). Rats were fasted overnight. Approximately 300 rag, wet wt, of tissue were placed in 25 ml Erlenmeyer flasks containing 5 ml of Krebs-Henseleit original Ringer bicarbonate (KRB) (Dawson, 1969) and 1 ml of a 10 mM glucose solution in water. Mebendazole was dissolved in DMSO (final DMSO concentration: 0.16 per cent). Incubation was carried out for 30 rain at 37°C under air. At the end of the incubation period the tissue was washed with KRB and homogenized (Ultra-Turrax) in water to a final volume of 3 mi. One millilitre of Ba(OH)z (1.8%) and 1 ml of ZnSO4 (2%) was added to 1 mi of the homogenate. After centrifugation, the glucose content of the supernatant was determined by the Glucostat method (Worthington Biochemical Corporation). E. Effect of mebendazole on the glycogen content Ascaris suum were incubated as described for the uptake studies with the exception that the glucose concentrations added to the incubation mixtures of the 24, 48 and 72 h experiments were 1-6, 2.66 and 3.7 x 10 -2 M respectively. At the end of the incubation periods the worms were dissected, the intestinal tract and muscle plus cuticle collected, weighed and digested in 6 and 20 ml K O H 30~o at 100°C for 30 rain. Glycogen was precipitated with ethanol and determined by the anthrone method as described in a previous study (Van den Bossche et aL, 1969).

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EFFECTS OF MEBENDAZOLE ON

Ascaris

403

RESULTS

The distribution of mebendazole and/or metabolites, in the different organs and pseudoeoelomic fluid of Ascaris, 24 h after incubation of the worms in a glucose salt medium (medium B), is shown in Table 1. Highest specific activity (counts/rain per g wet wt) was encountered in the alimentary system (pharynx plus intestine). However highest total activity was found in the pseudocoelomic fluid and muscle.

TABLE I - - D I S T R I B U T I O N OF 14C-LABELLED MEBENDAZOLE IN THE DIFFERENT ORGANS OF A. s////m

Radioactivity* Specific activity (counts/min per g tissue)

Organ Cuticle -k Muscle Intestine Reproductive system Pseudocoelomic fluid

4994 10052 2687 9382

4± 44-

817 3005 517 3108

Total activity (counts/min per total wt) 16874 3843 4247 18965

4- 29 4- 1354 -t- 361 4- 7649

* Mean value of 4 determinations 4-S.D.

The results shown in Table 2 indicate that mebendazole had no effect on the glycogen content of the muscle cells when glucose was omitted from the medium. Incubation of Ascaris in the presence of 3.4 × 10-6 M mebendazole and of glucose resulted after 48 h in a significant glycogen depletion. Increasing the dose twice, affected the glycogen content already after 24 h incubation. Neither the addition of 6"8 × 10-6M mebendazole nor incubation for 72 h in the presence of 3-4 × 10-6 M mebendazole affected the glycogen content of the intestinal cells (Table 3).

TABLE 2--EFFECT

Additions

Glucose +

OF MEBENDAZOLE ON THE GLYCOGEN CONTENT OF

Mebendazole ( × 10 -6 M)

Ascari$ MUSCLE*

Glycogen'[" (mg/g muscle) 1st day

2nd day

3rd day

0 3"4

25"35 ± 13"62 (9) 21"56 -+- 4"06 (9)i

19"26 4- 5"43 (4) 21"66 4- 8"12 (4)x

19-79 4- 5"10 (4) 18"22 4- 3"47 (4)I

0 3.4 6.8

33.39 4- 8.69 (8) 29.62 4- 7.40 (9)t 16-99 4- 7-39 (4)s

31.23 -4- 4.28 (4) 13.93 4- 5.41 (4)a

22.38 4- 5-44 (4) 13.18 4- 2-32 (4)z

were incubated for 24, 48 or 72 h in a medium containing DMSO or mebendazole. t Mean value :[:S.D. followed by the number of determinations in brackets. Px (Mann-Whitney U-test) > 0.05; P2 < 0-05; P3 ~< 0-01. + Details are presented in Materials and Methods: E. * Ascaris

FARA31.~"-I

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TABLE 3~EFFECT OF MEBENDAZOLEON THE GLYCOGEN CONTENT OF Ascaris INTESTINE*

Glycogen (mg/g intestine) Mebendazole Additions

Glucose

M)

1st day

2nd day

3rd day

0 3'4

2-36 4- 1"85 (9) 2.58 4- 0'93 (9)

2"25 4- 0"80 (4) 2"05 4- 1'35 (4)

1"08 4- 0"99 (4) 0"66 ± 0"23 (4)

0 3.4 6.8

2.74 4- 2.80 (7) 2-36 4- 1.98 (9) 3.39 ± 2.04 (4)

3.67 4- 1.09 (4) 1.79 q- 0.75 (4)

2-80 4- 1.50 (4) 1.57 4- 1-02 (4)

( x 10 - e

* Details are presented in the legend to Table 2.

The effect of mebendazole upon the glucose, 3-O-methylglucose and fructose uptake by Ascaris is presented in Table 4. The results show that the addition of glucose to the medium

resulted after 24 h in a significant increase (P = 0.008) in the 3-O-methylglucose uptake. Mebendazole significantly decreased the uptake of both glucose and 3-O-methylglucose. Although the uptake and/or utilization of fructose is also inhibited by mebendazole, no statistically significant change in the uptake of this hexose was observed when glucose was added.

TABLE 4 - - - E n a c t OF MEBENDAZOLEON THE GLUCOSE, 3-O-METHYLGLUCO~E AND FRUCTOSE UPTAKE

BY A. suum Uptake (t,moles/24 h per g worm)* Additions DMSO Mebendazolet D M S O + glucose++

Mebendazolet + glucose +

Glucose

3-O-methylglucose

Fructose

--

0"33 4- 0-13 (7) 0.15 4- 0-06 (7)s

3.09 4- 1"43 (7) 1.15 4- 0.85 (8)2

0.77 4- 0.16 (5) 0.16 5:0.03 (6)3

2.22 4- 0.07 (6) 0.63 4- 0.37 (8)3

-161.38 4- 61.27 (90) 74.69 4- 33.13 (84)

* Mean value -4-S.D. followed by the number of determinations in brackets. P2 < 0.05; P3 ~< 0.01. t Mebendazole: 3.4 x 10 - e M. ++Glucose: 1"6 -4- 10 -2 M.

Mebendazole also inhibited the uptake of proline, glycine and methionine in either the presence or absence of glucose (Table 5). The results presented in Table 6 show that the uptake of palmitic acid in the presence of glucose is about three times that observed in the absence of it. Mebendazole significantly decreased the uptake and/or transport of this fatty acid when glucose was present. However no statistical significant difference was obtained when glucose was omitted from the medium. The results shown in Table 7 indicate that mebendazole did not affect the glucose uptake by rin~s of everted rat small intestine even at a concentration up to 3"4 × 10 -s M.

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r,r r E c r s OF MEBENDAZOLE ON

TABLE5---EFrr~cr oF

Ascaris

405

MEBENDAZOLE ON THE PROLINE~ GLYCINE AND METHIONINE UPTAKE BY A . $////m*

Uptake ~moles/24 h per g worm) Additions

Proline

Glycine

Methionine

DMSO Mebendazole

4.68 4- 1.70 (10) 0"53 4- 0.44 (9)3

3"51 -4- 1.90 (13) 0.93 4- 0-32 (13)3

3.04 4- 1.02 (10) 0.37 4- 0.10 (ll)a

DMSO + Glucose Mebendazole + glucose

8.95 4- 3.93 (13) 0.72 4- 0-34 (10)a

7"62 -4- 4"86 (12) 2"41 4- 1"21 (14)a

9.37 4- 3.30 (9) 1.57 4- 1.01 (IDa

* Details are presented in the legend to Table 4.

TABLE 6--EFFECT OF MEBENDAZOLE ON THE PALMITIC ACID UPTAKE BY A. Sllllm*

Palmitic acid uptake (pmoles/24 h per g worm)

Additions

Pt

DMSO Mebendazole

0.24 -4- 0.11 (3) 0.22 4- 0.10 (3)

0.87

DMSO + glucose Mebendazole + glucose

0.74 4- 0.40 (7) 0.22 4- 0.05 (3)

0.0039

* Details are presented in the legend to Table 4. 1"Mann-Whitney U-test. TABLE 7 - - U P T A K E OF D-GLUCOSE BY RINGS OF RAT SMALL INTESTINE

Mebendazole

Glucose uptake*

(× 10-s M)

~,g/30 min per g tissue

0 0.34 3.40

52.5 4- 7.0 (6) 58.1 4- 7.8 (3) 48-1 4- 8-2 (3)

* Mean value -4-S.D. followed by the number of determinations in brackets. DISCUSSION The foregoing experiments indicate that mebendazole induces an increased glycogen utilization in the Ascaris muscle only when glucose was added to the incubation medium. These results are compatible with the previously reported hypothesis (Van den Bossche, 1972) that the mebendazole induced glycogen depletion is secondary to the inhibitory effect o f this drug on the glucose uptake. A n effect on the carbohydrate absorption b y A. suum and most other parasitic helminths, which live in environments with low oxygen tension, is disastrous since they depend for their energy supply almost entirely on c a r b o h y d r a t e catabolism (Saz, 1970). Therefore, the above mentioned effects o f mebendazole m a y be involved in the mechanism o f action o f this drug. N o effect was observed on the glycogen

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content of the intestinal cells. This may suggest that an inhibition of the absorptive processes in the intestinal tract results in a lower demand for energy, which leads to a decreased catabolism in the intestinal cells. Significant amounts of mebendazole and/or metabolites were found in the intestinal cells. This indicates that mebendazole is absorbed by these cells and can interfere with their absorption mechanisms. The experiments with rat small intestine indicate that mebendazole failed to exhibit an effect on the glucose uptake. This is in accord to the fact that mebendazole, administered orally at 2"5 or 10 mg per kg of body wt once per day for 13 weeks, failed to produce any effect on the glucose concentration in serum from beagle dogs. Furthermore, dietary administration of mebendazole to Wistar rats for 13 weeks at a dosage of 160 mg/100 g food did not affect the glucose content of the serum (Marsboom, 1973). Incubation of Ascaris in the presence of glucose resulted in an increased uptake of 3-O-methylglucose. This seems to indicate that the uptake and/or transport of this nonmetabolizable glucose derivative across the intestinal cells depends on energy. Similar results were obtained by Beames (1971). The absorption of proline, glycine and methionine also seems to be dependent upon energy since exogenous glucose significantly enhanced the rate of uptake of these amino acids. Mebendazole decreased the uptake of 3-O-methylglucose and of the amino acids whether or not glucose was added. This may indicate that the drug interferes with the passive--and glucose enhanced--transport mechanisms. The effect on the latter transport mechanism may be secondary to the mebendazole induced inhibition of the glucose uptake. Mebendazole also inhibits the uptake of fructose. However, in contrast with the 3-Omethylglucose uptake, the fructose uptake was not significantly affected by the addition of glucose. Our results disagree to those obtained by Beames (1971) in his studies on the isolated Ascaris intestine. The latter author found that the addition of glucose to the incubation mixture improved the fructose transport. The different results obtained may be the result of the different experimental conditions used. However our results are in accord to those obtained by Sanhueza et al. (1968) who failed to show a movement of hexoses from the luminal to the pseudocoelomic fluid against a concentration gradient. The brush border of Ascaris intestinal cells contains a low sucrase activity (Van den Bossche & Borgers, 1973), Furthermore Sanhueza et al. (lot. tit.) found that fructose was rapidly metabolized in the intestinal cells. Therefore it seems reasonable to assume that the fructose concentration, like that of glucose (Fairbairn & Passey, 1957), in the pseudocoelomic fluid is quite low. Thus it seems quite probable that with the intact worm, there may always be a favourable gradient for an energy independent transport of fructose. Beames & King (1972) found that palmitic acid moves into the intestinal cells via a passive process. However, energy from the catabolism of carbohydrate is required for the transport of fatty acid from the cell into the pseudocoelomic fluid. The results presented in this paper indicate that glucose added to the system facilitates the uptake and/or transport of palmitic acid complexed with a bile salt. Since mebendazole did not affect the fatty acid uptake when glucose was omitted from the medium it may be suggested that this drug has no effect on transport mechanisms which depend on energy obtained from the catabolism of endogenous sources, e.g. glycogen. In conclusion we can say that the experiments presented indicate that mebendazole inhibits in the absence and/or presence of glucose the uptake of low molecular weight nutrients by Ascaris. Ascaris depend for their energy supply almost entirely on glucose

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catabolism. Furthermore, only small amounts of amino acids, fructose and palmitic acid are absorbed as compared to the glucose uptake. Therefore, the mebendazole induced inhibition of the glucose uptake may be more involved in the mechanism of action of this drug than the inhibitory effect on the uptake of the other nutrients investigated here. REFERENCES BEAMES C. G. JR. 1971. Movement of hexoses across the midgut of Ascaris. Journal of Parasitology 57: 97-102. BEAMESC. G. JR. & KING G. A. 1972. Factors influencing the movement of materials across the intestine of Ascaris. In: Comparative Biochemistry o f Parasites (Edited by VAN DEN BOSSCHE, H.) pp. 275-282. Academic Press, New York. BUEDINO E., KMETEC E., SWARTZWELDERC., ABADm S. & SAZ H. J. 1961. Biochemical effects of dithiazanine on the canine whipworm, Trichuris vulpis. BiochemicalPharmacology 5:311-322. CRANE R. K. & MANDELSTAMP. 1960. Active transport of sugars by various preparations of hamster intestine. Biochimica et Biophysica Acta 45: 460--476. DAWSON R. M. C. 1969. Physiological media. In: Data for Biochemical Research (Edited by DAWSON, R. M. C., ELUOTT, D. C., ELLIOTT, W. H. & JONES, K. M.) p. 507. Oxford University Press, Oxford. FAXRBAmN D. & PASSEY R. F. 1957. The occurrence and distribution of trehalose and glycogen in the eggs and tissues of Ascaris lumbricoides, Experimental Parasitology 6: 566--574. HOFMANN A. F. 1963. The function of bile salts in fat absorption. The solvent properties of dilute miceUar solutions of conjugated bile salts. Biochemical Journal 89: 57-68. MAHIN D. T. & LOFBEROR. T. 1966. A simplified method of sample preparation for determination of tritium, carbon-14, or sulfur-35 in blood or tissue by liquid scintillation counting. Analytical Biochemistry 16: 500-509. MARSBOOM R. 1973. Toxicological studies on mebendazole. Toxicology and Applied Pharmacology (in press). SANHUEZA P., PALMA R., OBERHAUSER E., OREOO H., PARSONS D. S. & SALINAS A. 1968. Absorption of carbohydrates by intestine of Ascaris lumbricoides in vitro. Nature, Lond. 219: 1062-1063. SAZ H. J. 1970. Comparative energy metabolisms of some parasitic helminths. Journal of Parasitology 56: 634-642. VAN DEN BOSSCHE H. 1972. Biochemical effects of the anthelmintic drug mebendazole. In: Comparative Biochemistry of Parasites (Edited by VAN DEN BOSSCHE, H.) pp. 139-157. Academic Press, New York. VAN DEN BosscnE H. & BORGERS M. 1973. Subcellular distribution of digestive enzymes in Ascaris suum intestine. International Journal for Parasitology 3: 59-65. VAN DEN BOSSCHE H., VANPARUS O. F. J. &THIEr,rPONT D. 1969. Studies on the carbohydrate metabolism of third-stage Haemonchas contortus larvae. Life Sciences 8: 1047-1054. y o n BRAND T. 1972. Parasitenphysiologie p. 24. Gustav Fischer, Stuttgart.