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Glucose uptake in Oesophagostomum dentatum and the effect of oxfendazole
Veterinary Parasitology 80 (1998) 159±166
Glucose uptake in Oesophagostomum dentatum and the effect of oxfendazole Mads Bjelke Petersen*,1, Christian Friis Department of Pharmacology and Pathobiology, Royal Veterinary and Agricultural University, 13 BuÈlowsvej, DK-1870 Frederiksberg C, Denmark Received 24 February 1998; accepted 4 July 1998
Abstract The uptake of 14 C-glucose by adult Oesophagostomum dentatum was characterised. The uptake was a non-linear function of external glucose concentration. The maximum velocity of uptake (Vmax) was 0.964 nmol/100 mg dry weight (dw)/5 min, and the transport constant (Kt) was 10.02 mM. When phlorizin, phloretin and 3-O-methylglucose were tested for their effects on the uptake of 14 C-glucose, phloretin and 3-O-methylglucose produced significant inhibitions, indicating that the uptake was mediated and occurred by facilitated diffusion. Exposure of the worms to oxfendazole prior to incubation with 14 C-glucose did not affect the uptake of glucose. In another experiment worms were incubated with unlabelled glucose and the external glucose concentration was measured enzymatically. During a 7 h incubation period, the quantity of glucose remaining in the incubation medium of oxfendazole exposed worms was significantly greater than in the control group. It was concluded that oxfendazole did not influence the process of 14 C-glucose uptake, but might induce changes in the parasite leading to a reduced ability to deplete the incubation medium of glucose. # 1998 Elsevier Science B.V. All rights reserved. Keywords: Oesophagostomum dentatum; Glucose uptake; Oxfendazole
1. Introduction Benzimidazoles are widely used in veterinary medicine as broad spectrum anthelmintics with high efficacy against most nematodes (see Campbell, 1990). The mechanism of action of benzimidazoles involves binding to parasite tubulin (Lacey and * Corresponding author. Tel.: +45-44-94-58-88 ext. 2875; fax: +45-44-94-74-88; e-mail: [email protected] 1 Present address: Leo Pharmaceutical Products, 55 Industriparken, DK-2750 Ballerup, Denmark 0304-4017/98/$ ± see front matter # 1998 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 4 0 1 7 ( 9 8 ) 0 0 1 9 8 - 8
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Prichard, 1986). This leads to an inhibition of the polymerisation of tubulin into microtubules and to multiple biochemical changes and a general disruption of basic cell functions in the parasite (Lacey, 1988). In Ascaris suum, Trichinella spiralis and Trichuris globulosa exposure to benzimidazoles lead to an inhibition of glucose uptake (De Nollin and Van den Bossche, 1973; Van den Bossche and De Nollin, 1973; Jasra et al., 1990; Sarwal et al., 1992). In contrast, no effects of benzimidazoles on the glucose uptake in Haemonchus contortus and Trichostrongylus colubriformis were observed (Rew et al., 1982; Sangster and Prichard, 1984). The uptake of glucose in nematodes is reported to take place by mediated processes, which are comparable to those of the mammalian organism. These processes include active sodium dependent transport and facilitated diffusion (Sanhueza et al., 1968; Rutherford and Webster, 1974; Sarwal et al., 1992). The nodular worm, Oesophagostomum dentatum is a common parasite in the large intestine of the pig and no information of in vitro effects of benzimidazoles in this nematode has been available. The present study was designed to initially characterise the mechanism of glucose uptake by O. dentatum and subsequently to investigate possible effects on the uptake of glucose by the benzimidazole, oxfendazole. 2. Materials and methods 2.1. Nematode For the availability of adult O. dentatum, female pigs (25±30 kg) fed ground barley and mineral supplement, were infected with approximately 6000 third-stage larvae by stomach tube. The isolate of O. dentatum used in these studies was originally described by Roepstorff et al. (1987) and was considered to be susceptible to all anthelmintics. The presence of adult worms was indicated by the appearance of helminth eggs in faeces 3±4 weeks after the infection. Egg counts were determined by a modified McMaster technique. Between 28 and 35 days after the infection the pigs were euthanised by captive bolt pistol stunning and bled with the worms isolated from the intestinal contents using an agar-gel technique described by Slotved et al. (1996). 2.2. Uptake of
14
C-glucose
Isolated, clean worms were transferred to 25 mM tris±(hydroxymethyl)-aminomethane±HCl buffered Krebs-Ringer (KRT) saline (143 mM NaCl, 4.80 mM KCl, 2.58 mM CaCl2, 2.46 mM Mg2SO4) at pH 7.4 (see McCracken and Taylor, 1983) fortified with antibiotics (sodium-benzylpenicillin 200 iu/ml and dihydrostreptomycinsulphate 130 mg/ml) and preincubated in a 378C shaking water bath (60 cycles/min) for 6, 12 or 24 h. Groups of worms (40±50 worms, 70±90 mg wet weight) were then placed in 25 ml erlenmeyer flasks containing 3 ml KRT and, in some cases, phlorizin, phloretin or 3-Omethylglucose as inhibitors of glucose uptake. Two-hundred ml of KRT containing 2.96 kBq 14 C-glucose (Du Pont, USA) and unlabelled glucose at 16 final concentration were then added to each flask and worms were incubated with glucose for up to 40 min. The final glucose concentration in all incubations was 10 mM except for the studies of
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concentration-dependent uptake of glucose. In these studies the final glucose concentrations ranged from 1 to 1000 mM. In sodium-free incubations NaCl in the medium was replaced with KCl and potassium-benzylpenicillin was substituted for sodiumbenzylpenicillin. In the studies of the effect of oxfendazole on the uptake of glucose stock solutions of oxfendazole were prepared in dimethylsulphoxide (DMSO) with the final concentration of DMSO in the incubation media being 0.1%. Control incubations similarly contained 0.1% DMSO. For these studies the worms were preincubated with oxfendazole for 24 h prior to the incubation with glucose. Incubations were terminated by rinsing worms in two 5 ml changes of ice-cold KRT whereafter they were transferred to pre-weighed plastic scintillation vials and were dried under phosphorous pentoxide. The vials were weighed again and digested with Soluene-350 (Packard, The Netherlands) at 508C for 3 h. Hionic Fluor counting solution (Packard) was added and 14 C content determined by scintillation counting (LKB-Wallac 1217). The 14 C-activity in samples of the incubation medium was similarly measured by liquid scintillation counting using Optiphase `HiSafe' 2 counting solution (Wallac, Finland). 2.3. Glucose depletion Groups of worms (approximately 350 mg wet weight) were incubated at 378C for 18 h with or without oxfendazole at 10 mM. They were then transferred to 5 ml KRT containing 1 mM glucose in 25 ml flasks and incubated in a shaking water bath for 7 h (378C, 60 cycles/min). The glucose concentration of the incubation medium was monitored by taking samples every 60 min. The samples were immediately frozen (ÿ188C). After centrifugation (1000g for 10 min) the glucose concentration in the samples was measured by the hexokinase method (Gluco-quant1, BoehringerMannheim). 2.4. Data analysis For the estimation of the maximum velocity of uptake (Vmax) and the transport constant (Kt) concentration-dependent uptake of 14 C-glucose was fitted to the following equation: Y Vmax X= Kt X by non-linear regression analysis using the SAS1 System (SAS, 1988). In this equation Y represents the uptake of 14 C-glucose at a given concentration of glucose, represented by X. For comparison of two means a paired t-test was used. For comparison of more than two means two-way analysis of variance was used. Differences between groups were identified by Tukey's HSD test with a level of significance of 5%. 3. Results 3.1.
14
C-glucose uptake
The uptake of 14 C-glucose after preincubation times of 6, 12 and 24 h is presented in Fig. 1. Significantly more 14 C-glucose was taken up by O. dentatum after a
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Fig. 1. Uptake of 14 C-glucose by O. dentatum after preincubation in glucose free medium for 6, 12 and 24 h. Worms were incubated with 10 mM glucose for 20 min. Groups bearing different superscripts are significantly different (p<0.01). MeanS.D., n4. dwdry weight.
pre-incubation period of 24 h than after 6 and 12 h. A time-curve of the uptake of 14 Cglucose showed that the uptake was linear for at least 20 min (data not shown). The uptake of 14 C-glucose by O. dentatum was a non-linear function of the external glucose concentration (Fig. 2). At concentrations of glucose above 100 mM the increase in 14 Cglucose uptake was linear. The slope of this line was considered to represent uptake by diffusion. Mediated uptake of 14 C-glucose was then determined by subtracting diffusion
Fig. 2. Concentration-dependent uptake of 14 C-glucose by O. dentatum after preincubation in glucose free medium for 24 h. &: saturable component of 14 C-glucose uptake. &: diffusion component of 14 C-glucose uptake. dwdry weight.
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Fig. 3. Uptake of 14 C-glucose in O. dentatum in the presence of various inhibitors of glucose uptake. Preincubation time in glucose free medium was 24 h. Incubation time with glucose and inhibitors was 20 min. Concentration of phlorizin and phloretin was 50 mM. The concentration of 3-O-methylglucose was 10 mM. Groups bearing different superscripts are significantly different (p<0.05). MeanS.D., n4. dwdry weight.
from the observed uptake. Four individual determinations of 14 C-glucose absorption kinetics gave a Vmax of 0.9640.201 nmol/100 mg dry weight (dw)/5 min and a Kt of 10.022.25 mM (meanS.D.). When phloretin, phlorizin (both 50 mM) and 3-Omethylglucose (10 mM) were tested for their effects on the uptake of 14 C-glucose, phloretin and 3-O-methylglucose produced significant inhibitions (Fig. 3). Substituting K for Na in the incubation medium had no effect on the uptake of 14 C-glucose. The uptake of 14 C-glucose was not reduced after preincubation of worms with oxfendazole (Table 1). The worms that had been exposed to oxfendazole were less motile than control worms, but were intact and still living. 3.2. Glucose depletion The glucose concentrations in the incubation media at sampling times 1±7 h are presented in Fig. 4. At all sampling times the glucose concentration was higher in the oxfendazole treated group. As above, oxfendazole exposed worms appeared less motile than control worms, but were intact and still living. Table 1 Uptake of 14 C-glucose by O. dentatum after exposure to 10 mM oxfendazole for 24 h. Incubation time with glucose was 20 min. MeanS.D., n4 Group
Glucose uptake (nmol/100 mg dry weight)
Control 0.1% DMSOa 10 mM oxfendazole
3.401.24b 3.491.64b 3.682.47b
a
Solvent for oxfendazole. No significant differences between groups.
b
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Fig. 4. Quantity of glucose remaining in incubation medium after exposure of O. dentatum to 10 mM oxfendazole. *: p<0.05, **: p<0.01. MeanS.D., n4.
4. Discussion The concentration-dependent uptake of 14 C-glucose reported in the present study indicated the presence of a mediated system for glucose transport in O. dentatum. The mediated nature of the process was confirmed by the inhibition of 14 C-glucose uptake by phloretin and by 3-O-methylglucose. Phloretin inhibits facilitated diffusion of glucose (LeFevre, 1959). No inhibition was observed when worms were incubated with phlorizin, which is an inhibitor of active, sodium coupled transport of glucose (Hopfer et al., 1975). In accordance with this, the uptake of 14 C-glucose was unaffected by the absence of sodium. In contrast the uptake of glucose by T. globulosa was sodium-dependent indicating an active uptake mechanism (Sarwal et al., 1992). In Mermis nigrescens the uptake of glucose was sensitive to phloretin, indicating facilitated diffusion of glucose (Rutherford and Webster, 1974). The Vmax for glucose in T. globulosa was 9.25 mmol/ 100 mg dw/2 min (Sarwal et al., 1992), whereas the amounts of glucose delivered by the system reported in the present study are small having a Vmax of less than 1 nmol/100 mg dw/5 min. This indicates the presence of a high affinity, low capacity glucose transport system in O. dentatum. The natural habitat of O. dentatum is the lumen of the distal part of the colon of the pig (Jacobs, 1967) and while the concentration of glucose at this site is not known it is assumed to be very low in the highly reductive environment. Under these circumstances a high affinity system may function as a scavenger of available glucose in the environment rather than a primary source of nutrient. The site of absorption of glucose in O. dentatum is not known. In M. nigrescens the mediated phloretin sensitive uptake of glucose was transcuticular (Rutherford and Webster, 1974) and studies by Geary et al. (1993) indicated that in H. contortus absorption of glucose occurred through the cuticle. In A. suum active, phlorizin sensitive glucose uptake was located in the intestinal wall (Sanhueza et al., 1968), but the cuticle of this nematode is permeable to glucose (Fleming and Fetterer, 1984).
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An increase in the uptake of glucose was induced by preincubating O. dentatum in glucose free medium for 24 h prior to incubation with 14 C-glucose. This was probably caused by a depletion of endogenous energy reserves. This could be useful in studies of the effect of oxfendazole on the uptake of 14 C-glucose, because the activity of benzimidazoles is time-dependent (Prichard et al., 1978; Lacey, 1988). However, the uptake of 14 C-glucose of oxfendazole exposed worms was not different from the uptake of 14 C-glucose of control worms. On the other hand, when worms were incubated with 1 mM glucose for 7 h, the depletion of glucose from the incubation medium for oxfendazole exposed worms was lower than for control worms. With the mediated system reported above operating at Vmax for 7 h the total amount of glucose delivered would be approximately 80 nmol/100 mg dw. However, the total amount of glucose consumed during the 7 h period was almost 5 mmol. Based on a dry matter content of O. dentatum of 27% (M.B. Petersen, unpublished observations) the worms in a flask (350 mg wet weight) removed glucose at an average velocity of 60 nmol/100 mg dw/5 min over the 7 h period. This large component greatly exceeds the capacity of the mediated system and possibly results from diffusion. The lowered depletion of glucose in drug exposed worms was probably a reflection of the worms being of a poor condition. The worms that had been exposed to oxfendazole appeared less motile than control worms, but were intact and still living. A general disruption of basic cell functions, however, may be the consequence of exposure to benzimidazoles and the effects of oxfendazole observed in this study may be a reflection of such processes. Acknowledgements The authors wish to thank Dr. Henrik Bjùrn of the National Veterinary Laboratory, Denmark for assistance with parasitological techniques.
References Campbell, W.C., 1990. Benzimidazoles: veterinary uses. Parasitol. Today 6, 130±133. De Nollin, S., Van den Bossche, H., 1973. Biochemical effects of mebendazole on Trichinella spiralis larvae. J. Parasitol. 59, 970±976. Fleming, M.W., Fetterer, R.H., 1984. Ascaris suum: continuous perfusion of the pseudocoelom and nutrient absorption. Exp. Parasitol. 57, 142±148. Geary, T.G., Sims, S.M., Thomas, E.M., Vanover, L., Davis, J.P., Winterrowd, C.A., Klein, R.D., Ho, N.F.H., Thompson, D.P., 1993. Haemonchus contortus: ivermectin-induced paralysis of the pharynx. Exp. Parasitol. 77, 88±96. Hopfer, U., Sigrist-Nelson, K., Murer, H., 1975. Intestinal sugar transport: studies with isolated plasma membranes. Ann. New York Acad. Sci. 264, 414±426. Jacobs, D.E., 1967. The linear distribution of two Oesophagostomum species in the intestine of the pig. Acta Vet. Scand. 8, 287±289. Jasra, N., Sanyal, S.N., Khera, S., 1990. Effect of thiabendazole and fenbendazole on glucose uptake and carbohydrate metabolism in Trichuris globulosa. Vet. Parasitol. 35, 201±209. Lacey, E., 1988. The role of the cytoskeletal protein, tubulin, in the mode of action and mechanism of drug resistance to benzimidazoles. Int. J. Parasitol. 18, 885±936.
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Lacey, E., Prichard, R.K., 1986. Interactions of benzimidazoles (BZ) with tubulin from BZ-sensitive and BZresistant isolates of Haemonchus contortus. Mol. Biochem. Parasitol. 19, 171±181. LeFevre, P.G., 1959. Molecular structural factors in competitive inhibition of sugar transport. Science 130, 104± 105. McCracken, R.O., Taylor, D.D., 1983. Biochemical effects of fenbendazole on Hymenolepis diminuta in vivo. Int. J. Parasitol. 13, 267±272. Prichard, R.K., Hennessy, D.R., Steel, J.W., 1978. Prolonged administration: a new concept for increasing the spectrum and effectiveness of anthelmintics. Vet. Parasitol. 4, 309±315. Rew, R.S., Smith, C., Colglazier, M.L., 1982. Glucose metabolism of Haemonchus contortus adults: effects of thiabendazole on susceptible versus resistant strain. J. Parasitol. 68, 845±850. Roepstorff, A., Bjùrn, H., Nansen, P., 1987. Resistance of Oesophagostomum spp. in pigs to pyrantel citrate. Vet. Parasitol. 24, 229±239. Rutherford, T.A., Webster, J.M., 1974. Transcuticular uptake of glucose by the entomophilic nematode, Mermis nigrescens. J. Parasitol. 60, 804±808. Sangster, N.C., Prichard, R.K., 1984. Uptake of thiabendazole and its effects on glucose uptake and carbohydrate levels in the thiabendazole-resistant and susceptible Trichostrongylus colubriformis. Int. J. Parasitol. 14, 121±126. Sanhueza, P., Palma, R., Oberhauser, E., Orrego, H., Parsons, D.S., Salinas, A., 1968. Absorption of carbohydrates by intestine of Ascaris lumbricoides in vitro. Nature 219, 1062±1063. Sarwal, R., Sanyal, S.N., Khera, S., 1992. Effect of benzimidazole drugs on the uptake of low molecular weight nutrients in Trichuris globulosa. Int. J. Parasitol. 22, 9±14. SAS Institute Inc., 1988. SAS/STAT1 User's Guide, Release 6.03 ed. Cary, NC: SAS Institute Inc., pp. 549±640. Slotved, H.-C., Barnes, E.H., Bjùrn, H., Christensen, C.M., Eriksen, L., Roepstorff, A., Nansen, P., 1996. Recovery of Oesophagostomum dentatum from pigs by isolation of parasites migrating from large intestinal contents embedded in agar-gel. Vet. Parasitol. 63, 237±245. Van den Bossche, H., De Nollin, S., 1973. Effects of mebendazole on the absorption of low molecular weight nutrients by Ascaris suum. Int. J. Parasitol. 3, 401±407.
Glucose uptake in Oesophagostomum dentatum and the effect of oxfendazole Mads Bjelke Petersen*,1, Christian Friis Department of Pharmacology and Pathobiology, Royal Veterinary and Agricultural University, 13 BuÈlowsvej, DK-1870 Frederiksberg C, Denmark Received 24 February 1998; accepted 4 July 1998
Abstract The uptake of 14 C-glucose by adult Oesophagostomum dentatum was characterised. The uptake was a non-linear function of external glucose concentration. The maximum velocity of uptake (Vmax) was 0.964 nmol/100 mg dry weight (dw)/5 min, and the transport constant (Kt) was 10.02 mM. When phlorizin, phloretin and 3-O-methylglucose were tested for their effects on the uptake of 14 C-glucose, phloretin and 3-O-methylglucose produced significant inhibitions, indicating that the uptake was mediated and occurred by facilitated diffusion. Exposure of the worms to oxfendazole prior to incubation with 14 C-glucose did not affect the uptake of glucose. In another experiment worms were incubated with unlabelled glucose and the external glucose concentration was measured enzymatically. During a 7 h incubation period, the quantity of glucose remaining in the incubation medium of oxfendazole exposed worms was significantly greater than in the control group. It was concluded that oxfendazole did not influence the process of 14 C-glucose uptake, but might induce changes in the parasite leading to a reduced ability to deplete the incubation medium of glucose. # 1998 Elsevier Science B.V. All rights reserved. Keywords: Oesophagostomum dentatum; Glucose uptake; Oxfendazole
1. Introduction Benzimidazoles are widely used in veterinary medicine as broad spectrum anthelmintics with high efficacy against most nematodes (see Campbell, 1990). The mechanism of action of benzimidazoles involves binding to parasite tubulin (Lacey and * Corresponding author. Tel.: +45-44-94-58-88 ext. 2875; fax: +45-44-94-74-88; e-mail: [email protected] 1 Present address: Leo Pharmaceutical Products, 55 Industriparken, DK-2750 Ballerup, Denmark 0304-4017/98/$ ± see front matter # 1998 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 4 0 1 7 ( 9 8 ) 0 0 1 9 8 - 8
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Prichard, 1986). This leads to an inhibition of the polymerisation of tubulin into microtubules and to multiple biochemical changes and a general disruption of basic cell functions in the parasite (Lacey, 1988). In Ascaris suum, Trichinella spiralis and Trichuris globulosa exposure to benzimidazoles lead to an inhibition of glucose uptake (De Nollin and Van den Bossche, 1973; Van den Bossche and De Nollin, 1973; Jasra et al., 1990; Sarwal et al., 1992). In contrast, no effects of benzimidazoles on the glucose uptake in Haemonchus contortus and Trichostrongylus colubriformis were observed (Rew et al., 1982; Sangster and Prichard, 1984). The uptake of glucose in nematodes is reported to take place by mediated processes, which are comparable to those of the mammalian organism. These processes include active sodium dependent transport and facilitated diffusion (Sanhueza et al., 1968; Rutherford and Webster, 1974; Sarwal et al., 1992). The nodular worm, Oesophagostomum dentatum is a common parasite in the large intestine of the pig and no information of in vitro effects of benzimidazoles in this nematode has been available. The present study was designed to initially characterise the mechanism of glucose uptake by O. dentatum and subsequently to investigate possible effects on the uptake of glucose by the benzimidazole, oxfendazole. 2. Materials and methods 2.1. Nematode For the availability of adult O. dentatum, female pigs (25±30 kg) fed ground barley and mineral supplement, were infected with approximately 6000 third-stage larvae by stomach tube. The isolate of O. dentatum used in these studies was originally described by Roepstorff et al. (1987) and was considered to be susceptible to all anthelmintics. The presence of adult worms was indicated by the appearance of helminth eggs in faeces 3±4 weeks after the infection. Egg counts were determined by a modified McMaster technique. Between 28 and 35 days after the infection the pigs were euthanised by captive bolt pistol stunning and bled with the worms isolated from the intestinal contents using an agar-gel technique described by Slotved et al. (1996). 2.2. Uptake of
14
C-glucose
Isolated, clean worms were transferred to 25 mM tris±(hydroxymethyl)-aminomethane±HCl buffered Krebs-Ringer (KRT) saline (143 mM NaCl, 4.80 mM KCl, 2.58 mM CaCl2, 2.46 mM Mg2SO4) at pH 7.4 (see McCracken and Taylor, 1983) fortified with antibiotics (sodium-benzylpenicillin 200 iu/ml and dihydrostreptomycinsulphate 130 mg/ml) and preincubated in a 378C shaking water bath (60 cycles/min) for 6, 12 or 24 h. Groups of worms (40±50 worms, 70±90 mg wet weight) were then placed in 25 ml erlenmeyer flasks containing 3 ml KRT and, in some cases, phlorizin, phloretin or 3-Omethylglucose as inhibitors of glucose uptake. Two-hundred ml of KRT containing 2.96 kBq 14 C-glucose (Du Pont, USA) and unlabelled glucose at 16 final concentration were then added to each flask and worms were incubated with glucose for up to 40 min. The final glucose concentration in all incubations was 10 mM except for the studies of
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concentration-dependent uptake of glucose. In these studies the final glucose concentrations ranged from 1 to 1000 mM. In sodium-free incubations NaCl in the medium was replaced with KCl and potassium-benzylpenicillin was substituted for sodiumbenzylpenicillin. In the studies of the effect of oxfendazole on the uptake of glucose stock solutions of oxfendazole were prepared in dimethylsulphoxide (DMSO) with the final concentration of DMSO in the incubation media being 0.1%. Control incubations similarly contained 0.1% DMSO. For these studies the worms were preincubated with oxfendazole for 24 h prior to the incubation with glucose. Incubations were terminated by rinsing worms in two 5 ml changes of ice-cold KRT whereafter they were transferred to pre-weighed plastic scintillation vials and were dried under phosphorous pentoxide. The vials were weighed again and digested with Soluene-350 (Packard, The Netherlands) at 508C for 3 h. Hionic Fluor counting solution (Packard) was added and 14 C content determined by scintillation counting (LKB-Wallac 1217). The 14 C-activity in samples of the incubation medium was similarly measured by liquid scintillation counting using Optiphase `HiSafe' 2 counting solution (Wallac, Finland). 2.3. Glucose depletion Groups of worms (approximately 350 mg wet weight) were incubated at 378C for 18 h with or without oxfendazole at 10 mM. They were then transferred to 5 ml KRT containing 1 mM glucose in 25 ml flasks and incubated in a shaking water bath for 7 h (378C, 60 cycles/min). The glucose concentration of the incubation medium was monitored by taking samples every 60 min. The samples were immediately frozen (ÿ188C). After centrifugation (1000g for 10 min) the glucose concentration in the samples was measured by the hexokinase method (Gluco-quant1, BoehringerMannheim). 2.4. Data analysis For the estimation of the maximum velocity of uptake (Vmax) and the transport constant (Kt) concentration-dependent uptake of 14 C-glucose was fitted to the following equation: Y Vmax X= Kt X by non-linear regression analysis using the SAS1 System (SAS, 1988). In this equation Y represents the uptake of 14 C-glucose at a given concentration of glucose, represented by X. For comparison of two means a paired t-test was used. For comparison of more than two means two-way analysis of variance was used. Differences between groups were identified by Tukey's HSD test with a level of significance of 5%. 3. Results 3.1.
14
C-glucose uptake
The uptake of 14 C-glucose after preincubation times of 6, 12 and 24 h is presented in Fig. 1. Significantly more 14 C-glucose was taken up by O. dentatum after a
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Fig. 1. Uptake of 14 C-glucose by O. dentatum after preincubation in glucose free medium for 6, 12 and 24 h. Worms were incubated with 10 mM glucose for 20 min. Groups bearing different superscripts are significantly different (p<0.01). MeanS.D., n4. dwdry weight.
pre-incubation period of 24 h than after 6 and 12 h. A time-curve of the uptake of 14 Cglucose showed that the uptake was linear for at least 20 min (data not shown). The uptake of 14 C-glucose by O. dentatum was a non-linear function of the external glucose concentration (Fig. 2). At concentrations of glucose above 100 mM the increase in 14 Cglucose uptake was linear. The slope of this line was considered to represent uptake by diffusion. Mediated uptake of 14 C-glucose was then determined by subtracting diffusion
Fig. 2. Concentration-dependent uptake of 14 C-glucose by O. dentatum after preincubation in glucose free medium for 24 h. &: saturable component of 14 C-glucose uptake. &: diffusion component of 14 C-glucose uptake. dwdry weight.
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Fig. 3. Uptake of 14 C-glucose in O. dentatum in the presence of various inhibitors of glucose uptake. Preincubation time in glucose free medium was 24 h. Incubation time with glucose and inhibitors was 20 min. Concentration of phlorizin and phloretin was 50 mM. The concentration of 3-O-methylglucose was 10 mM. Groups bearing different superscripts are significantly different (p<0.05). MeanS.D., n4. dwdry weight.
from the observed uptake. Four individual determinations of 14 C-glucose absorption kinetics gave a Vmax of 0.9640.201 nmol/100 mg dry weight (dw)/5 min and a Kt of 10.022.25 mM (meanS.D.). When phloretin, phlorizin (both 50 mM) and 3-Omethylglucose (10 mM) were tested for their effects on the uptake of 14 C-glucose, phloretin and 3-O-methylglucose produced significant inhibitions (Fig. 3). Substituting K for Na in the incubation medium had no effect on the uptake of 14 C-glucose. The uptake of 14 C-glucose was not reduced after preincubation of worms with oxfendazole (Table 1). The worms that had been exposed to oxfendazole were less motile than control worms, but were intact and still living. 3.2. Glucose depletion The glucose concentrations in the incubation media at sampling times 1±7 h are presented in Fig. 4. At all sampling times the glucose concentration was higher in the oxfendazole treated group. As above, oxfendazole exposed worms appeared less motile than control worms, but were intact and still living. Table 1 Uptake of 14 C-glucose by O. dentatum after exposure to 10 mM oxfendazole for 24 h. Incubation time with glucose was 20 min. MeanS.D., n4 Group
Glucose uptake (nmol/100 mg dry weight)
Control 0.1% DMSOa 10 mM oxfendazole
3.401.24b 3.491.64b 3.682.47b
a
Solvent for oxfendazole. No significant differences between groups.
b
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Fig. 4. Quantity of glucose remaining in incubation medium after exposure of O. dentatum to 10 mM oxfendazole. *: p<0.05, **: p<0.01. MeanS.D., n4.
4. Discussion The concentration-dependent uptake of 14 C-glucose reported in the present study indicated the presence of a mediated system for glucose transport in O. dentatum. The mediated nature of the process was confirmed by the inhibition of 14 C-glucose uptake by phloretin and by 3-O-methylglucose. Phloretin inhibits facilitated diffusion of glucose (LeFevre, 1959). No inhibition was observed when worms were incubated with phlorizin, which is an inhibitor of active, sodium coupled transport of glucose (Hopfer et al., 1975). In accordance with this, the uptake of 14 C-glucose was unaffected by the absence of sodium. In contrast the uptake of glucose by T. globulosa was sodium-dependent indicating an active uptake mechanism (Sarwal et al., 1992). In Mermis nigrescens the uptake of glucose was sensitive to phloretin, indicating facilitated diffusion of glucose (Rutherford and Webster, 1974). The Vmax for glucose in T. globulosa was 9.25 mmol/ 100 mg dw/2 min (Sarwal et al., 1992), whereas the amounts of glucose delivered by the system reported in the present study are small having a Vmax of less than 1 nmol/100 mg dw/5 min. This indicates the presence of a high affinity, low capacity glucose transport system in O. dentatum. The natural habitat of O. dentatum is the lumen of the distal part of the colon of the pig (Jacobs, 1967) and while the concentration of glucose at this site is not known it is assumed to be very low in the highly reductive environment. Under these circumstances a high affinity system may function as a scavenger of available glucose in the environment rather than a primary source of nutrient. The site of absorption of glucose in O. dentatum is not known. In M. nigrescens the mediated phloretin sensitive uptake of glucose was transcuticular (Rutherford and Webster, 1974) and studies by Geary et al. (1993) indicated that in H. contortus absorption of glucose occurred through the cuticle. In A. suum active, phlorizin sensitive glucose uptake was located in the intestinal wall (Sanhueza et al., 1968), but the cuticle of this nematode is permeable to glucose (Fleming and Fetterer, 1984).
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An increase in the uptake of glucose was induced by preincubating O. dentatum in glucose free medium for 24 h prior to incubation with 14 C-glucose. This was probably caused by a depletion of endogenous energy reserves. This could be useful in studies of the effect of oxfendazole on the uptake of 14 C-glucose, because the activity of benzimidazoles is time-dependent (Prichard et al., 1978; Lacey, 1988). However, the uptake of 14 C-glucose of oxfendazole exposed worms was not different from the uptake of 14 C-glucose of control worms. On the other hand, when worms were incubated with 1 mM glucose for 7 h, the depletion of glucose from the incubation medium for oxfendazole exposed worms was lower than for control worms. With the mediated system reported above operating at Vmax for 7 h the total amount of glucose delivered would be approximately 80 nmol/100 mg dw. However, the total amount of glucose consumed during the 7 h period was almost 5 mmol. Based on a dry matter content of O. dentatum of 27% (M.B. Petersen, unpublished observations) the worms in a flask (350 mg wet weight) removed glucose at an average velocity of 60 nmol/100 mg dw/5 min over the 7 h period. This large component greatly exceeds the capacity of the mediated system and possibly results from diffusion. The lowered depletion of glucose in drug exposed worms was probably a reflection of the worms being of a poor condition. The worms that had been exposed to oxfendazole appeared less motile than control worms, but were intact and still living. A general disruption of basic cell functions, however, may be the consequence of exposure to benzimidazoles and the effects of oxfendazole observed in this study may be a reflection of such processes. Acknowledgements The authors wish to thank Dr. Henrik Bjùrn of the National Veterinary Laboratory, Denmark for assistance with parasitological techniques.
References Campbell, W.C., 1990. Benzimidazoles: veterinary uses. Parasitol. Today 6, 130±133. De Nollin, S., Van den Bossche, H., 1973. Biochemical effects of mebendazole on Trichinella spiralis larvae. J. Parasitol. 59, 970±976. Fleming, M.W., Fetterer, R.H., 1984. Ascaris suum: continuous perfusion of the pseudocoelom and nutrient absorption. Exp. Parasitol. 57, 142±148. Geary, T.G., Sims, S.M., Thomas, E.M., Vanover, L., Davis, J.P., Winterrowd, C.A., Klein, R.D., Ho, N.F.H., Thompson, D.P., 1993. Haemonchus contortus: ivermectin-induced paralysis of the pharynx. Exp. Parasitol. 77, 88±96. Hopfer, U., Sigrist-Nelson, K., Murer, H., 1975. Intestinal sugar transport: studies with isolated plasma membranes. Ann. New York Acad. Sci. 264, 414±426. Jacobs, D.E., 1967. The linear distribution of two Oesophagostomum species in the intestine of the pig. Acta Vet. Scand. 8, 287±289. Jasra, N., Sanyal, S.N., Khera, S., 1990. Effect of thiabendazole and fenbendazole on glucose uptake and carbohydrate metabolism in Trichuris globulosa. Vet. Parasitol. 35, 201±209. Lacey, E., 1988. The role of the cytoskeletal protein, tubulin, in the mode of action and mechanism of drug resistance to benzimidazoles. Int. J. Parasitol. 18, 885±936.
166
M.B. Petersen, C. Friis / Veterinary Parasitology 80 (1998) 159±166
Lacey, E., Prichard, R.K., 1986. Interactions of benzimidazoles (BZ) with tubulin from BZ-sensitive and BZresistant isolates of Haemonchus contortus. Mol. Biochem. Parasitol. 19, 171±181. LeFevre, P.G., 1959. Molecular structural factors in competitive inhibition of sugar transport. Science 130, 104± 105. McCracken, R.O., Taylor, D.D., 1983. Biochemical effects of fenbendazole on Hymenolepis diminuta in vivo. Int. J. Parasitol. 13, 267±272. Prichard, R.K., Hennessy, D.R., Steel, J.W., 1978. Prolonged administration: a new concept for increasing the spectrum and effectiveness of anthelmintics. Vet. Parasitol. 4, 309±315. Rew, R.S., Smith, C., Colglazier, M.L., 1982. Glucose metabolism of Haemonchus contortus adults: effects of thiabendazole on susceptible versus resistant strain. J. Parasitol. 68, 845±850. Roepstorff, A., Bjùrn, H., Nansen, P., 1987. Resistance of Oesophagostomum spp. in pigs to pyrantel citrate. Vet. Parasitol. 24, 229±239. Rutherford, T.A., Webster, J.M., 1974. Transcuticular uptake of glucose by the entomophilic nematode, Mermis nigrescens. J. Parasitol. 60, 804±808. Sangster, N.C., Prichard, R.K., 1984. Uptake of thiabendazole and its effects on glucose uptake and carbohydrate levels in the thiabendazole-resistant and susceptible Trichostrongylus colubriformis. Int. J. Parasitol. 14, 121±126. Sanhueza, P., Palma, R., Oberhauser, E., Orrego, H., Parsons, D.S., Salinas, A., 1968. Absorption of carbohydrates by intestine of Ascaris lumbricoides in vitro. Nature 219, 1062±1063. Sarwal, R., Sanyal, S.N., Khera, S., 1992. Effect of benzimidazole drugs on the uptake of low molecular weight nutrients in Trichuris globulosa. Int. J. Parasitol. 22, 9±14. SAS Institute Inc., 1988. SAS/STAT1 User's Guide, Release 6.03 ed. Cary, NC: SAS Institute Inc., pp. 549±640. Slotved, H.-C., Barnes, E.H., Bjùrn, H., Christensen, C.M., Eriksen, L., Roepstorff, A., Nansen, P., 1996. Recovery of Oesophagostomum dentatum from pigs by isolation of parasites migrating from large intestinal contents embedded in agar-gel. Vet. Parasitol. 63, 237±245. Van den Bossche, H., De Nollin, S., 1973. Effects of mebendazole on the absorption of low molecular weight nutrients by Ascaris suum. Int. J. Parasitol. 3, 401±407.