Hymenolepis diminuta: Effects of Amoscanate on energy metabolism and ultrastructure

EXPERIMENTAL

PARASITOLOGY

56, 55-69 (1983)

Hymenolepis diminuta: Effects of Amoscanate on Energy Metabolism and Ultrastructure NORMAN E NELSONANDHOWARD

J. SAZ~

Department of Biology, University of Notre Dame, Notre Dame, Indiana 46556, U.S.A. (Accepted for publication 1 March 1983) NELSON, N. E, AND SAZ, H. J. 1983. Hymenolepis diminuta: Effects of amoscanate on energy metabolism and ultrastructure. Experimental Parasitology 56, 55-69. Amoscanate possesses chemotherapeutic activity against schistosomes, and in higher doses against many other helminths including filariids and Hymenolepis diminuta. The primary mode of action of this compound is unknown. Effects of the drug on the carbohydrate metabolism as well as on the tegumental and nephridial epithelia of H. diminuta were examined. At various time intervals after administration of the drug to rats infected with H. diminuta, the parasites were recovered and incubated in glucose-salts medium for 90 min. Chemotherapy resulted in decreases in succinate, lactate, and acetate recoveries, while ATP levels dropped. In addition, glycogen levels were depressed in drug-treated worms which were homogenized immediately upon isolation. Glycogen synthase I activity was inhibited 16-61% in cestodes obtained from Amoscanate-treated animals and homogenized immediately, but returned to normal levels after incubation for 90 min in glucose-salts medium prior to homogenization and assay. Phosphorylase a activity was found to be 25-30% higher in preparations of worms from drug-treated rats, which correlates with the rapid depletion of glycogen in parasites exposed to the drug. However, in contrast with glycogen synthase activity, the elevation of phosphorylase a activity in H. diminuta exposed to the drug was not readily reversible. Attempts to demonstrate activity of the drug in vitro by incubating intact cestodes directly with Amoscanate were unsuccessful. Thin sections of parasites obtained from Amoscanatetreated rats and examined by transmission electron microscopy revealed surface alterations of the tegument and nephridial canals. Alterations included bleb formation and erosion of microtriches from the tegument, as well as disappearance of microvilli from nephridial canals. However, these effects became manifest only after 4 or more hr exposure of the rat to the drug. Biochemical effects, on the other hand, were significant after 3 hr exposure. INDEX DESCRIPTORS: Hymenolepis diminuta; Cestoda; Tegument; Microtriches; Nephridial canals; Drug effects; Amoscanate; Electron microscopy, transmission; Carbohydrate metabolism; Glycogen synthase; Phosphorylase.

INTRODUCTION

Amoscanate (4-isothiocyanato-4’-nitrodiphenylamine) possesses chemotherapeutic activity against schistosomes (Bueding et al. 1976). In considerably higher doses, amoscanate also is effective against a number of other helminths, including filariids, Hjmenolepis diminuta, Ascaris lumbricoides, Nematospiroides dubius, and hookworms (Striebel 1976; Saz et al. 1977; Middleton et al. 1979). Therapeutic effecI To whom requests for reprints should be addressed.

tiveness of the drug in human infections also has been reported (Sen 1976; Doshi et al. 1977). Amoscanate is of particular interest, since it appears to have two independent modes of action. Schistosomes and filariids respond to a single oral dose which results in the death of the parasites, but not until approximately 2 months later (Bueding et al. 1976; Saz et al. 1977). In contrast, most other parasites are removed within 24 to 48 hr. However, little is known of the modes of action of this drug regarding either the short- or long-term effects. It has been suggested that the drug may disrupt the os-

55 0014-4894183$3.00 Copyright 0 1983 by Academic Press, Inc. All rights of reproduction in my formreserved.

NELSON AND SAZ

56

motic balance resulting in impaired transport in schistosomes (Voge and Bueding 1980). In an effort to examine the short-term activity of the drug, the effects of Amoscanate on the carbohydrate metabolism and ultrastructure of H. diminuta were investigated. Systems evaluated included glucose uptake, glycolysis, glycogen synthesis, glycogenolysis, and motility of the adult worms. Changes in ultrastructure also were examined by means of transmission electron microscopy. Alterations in surface topography were studied by scanning electron microscopy. A preliminary report of the effects of amoscanate on the carbohydrate metabolism has been presented (Nelson and Saz 1981). MATERIALS AND METHODS Hymenolepis diminuta infections were maintained in male Lobund-Wistar rats. Animals were infected and worms were recovered from the intestines after 14 or more days according to the method of Fairbairn et ai. (1961). After washing in 0.85% saline, blotting, and weighing, worms were incubated for 90 min under an atmosphere of 95% N,:5% CO, at 38 C in Warburg vessels containing 5 ml of Krebs-Ringer bicarbonate with or without 17 mM glucose (Umbreit et al. 1964). Incubations were terminated by removing and freezing the parasites in 2-methylbutane cooled to - 150C with liquid N?. The incubation media were centrifuged, and stored at - 20 C. Frozen tissues were homogenized in 2 ml of 2% perchloric acid (PCA) and centrifuged at 23,000g for 20 min. The supernatant fractions which contained perchloric acid were neutralized with KOH, and the KClO, was removed by centrifugation. ATP, carbohydrate/protein ratios, as well as succinate and lactate levels were determined in this tissue supema tant. Although only low levels of succinate and lactate were found in the worm, the amounts determined were added to those found in the fermentation media to give the total of each acid. Protein was determined according to Lowry et al. (1951) employing bovine serum albumin as standard. Incubations in the presence of l+C]glucose were terminated by transferring the worms to 2 ml of boiling water for 10 min followed by homogenization. Aliquots of the homogenates were transferred to tubes which contained KOH such that the final concentrations in each vessel was 30% KOH. The mixture was then heated in a boiling water bath for 2 hr and glycogen was isolated from an aliquot according to Wang and Saz (1974). The remainder of each samnle was

neutralized with 50% perchloric acid and centrifuged at lO,OOOgfor 10 min. Supematants were stored at - 20 C prior to chromatography on a Dowex l-X8 (Cl-) column for the isolation of metabolic acids as described by Von Korff (1969). The elution gradient was formed by mixing 250 ml of 0.01 N HCl and 250 ml of distilled water in a Pharmacia GM-l gradient mixer. Radioactivity was assayed in a liquid scintillation spectrometer employing Aquasol (New England Nuclear Corp. Boston, MA, USA) as the scintillation medium. Quench corrections were made by means of internal standardization. Glycogen synthase (UDP-glucose: glycogen a-Cglycosyl transferase; EC 2.4.1.11) activity in worms was determined after homogenizing at 4 C in 5 vol of 0.05 M glycylglycine (pH 7.4) which contained 8 mM 2mercaptoethanol and 1 mM ethylenediamine tetraacetate (EDTA). The independent or I form of glycogen synthase was prepared according to Mied and Bueding (1979), and assayed by measuring the incorporation of UDP-[14C]glucose(Uridine-5’-diphosphate glucose) into glycogen, employing the filter paper technique of Thomas ef al. (1968). The assay mixture contained in a total volume of 200 ul, 20 mM N-2-hydroxyethanylpiperazine-N’-2-ethanesulfonic acid (Hepes), pH 7.5, 16 mM 2-mercaptoethanol, 2 mM EDTA, and 20 mM UDP-[14C]glucose (25,100 dpm/pmol). After a IO-min incubation period at 32 C, loo-p1 aliquots were spotted on 2 x 2-cm squares of ET 31 filter paper (Whatman) which then were placed in a beaker containing ice cold 66% ethanol (10 ml/paper) and gently stirred for 15 min. The ethanol was replaced with two rinses of 66% ethanol for 15 min each, followed by a 5-min rinse in acetone. The filter papers were oven dried, placed individually in scintillation vials containing 10 ml of Aquasol, and counted. Glycogen phosphorylase (a-l ,Cglucan: orthophosphate-glucosyl-transferase (EC 2.4.1.1)) activity was determined in worms which were homogenized at 4 C in 5 vol of 0.05 M glycylglycine (pH 7.4) which contained 8 mM 2-mercaptoethanol and 20 mM NaF. Homogenates were centrifuged at 10,OOOgfor 10 min at 2-4 C and the supematant fraction was used for assay. The assay method employed was that described by Bueding and Fisher (1970). For electron microscopy, 14-day-old worms were removed from infected rats 0, 1, 2, 3, 4, and 8 hr after treatment with a single oral dose of 100 mg/kg amoscanate. The worms were rinsed several times in 0.9% NaCl, and placed in 0.1 M cacodylate buffer, pH 7.4, which contained 3% glutaraldehyde. Proglottid segments 2 and 5 cm distal from the scolex were removed and allowed to remain in fixative for 22 hr at 4 C. After two washes in 0.1 M cacodylate, pH 7.4, the segments were posttixed for 2 hr in 1% 0~0,. The worm segments then were processed separately for either transmission or scanning electron microscopy. For scanning electron microscopy, the worm seg-

Hymenolepis diminuta:

EFFECTS

ments were dehydrated in a series of graded acetone washes of 50, 70, 90, 95, and twice in 100% acetone for 10 min each. After critical point drying in a Pelco Model H liquid CO1 critical point dryer, the specimens were mounted on metal stubs, gold evaporated, and coated with the aid of a Denton DV-515 vacuum evaporator, prior to examination with a Cambridge 600 Stereoscan scanning electron microscope. For transmission electron microscopy, the worm segments were cut into sections 1 mm in thickness and dehydrated for 10 min in each of a series of alcohol washes of 50,70,90, and 95%, followed by two washes in 100% ethanol. Plastic infiltration was initiated by two IO-min washes in propylene oxide, followed by 30 min in 1:l Epon-propylene oxide. The samples were placed in 100% epon for 14 hr at room temperature, transferred to fresh Epon, and polymerization was completed by heating at 60 C for 36 hr. The Epon blocks were sectioned (600-800 A) with a diamond knife on an LKB Ultratome III, placed on copper grids, and stained with uranyl acetate and lead citrate. The samples were viewed with a Hitachi H-600 transmission electron microscope. Lactate and glucose were analyzed according to Lowry et al. (1964) and Slein (1%3), respectively. Succinate was quantitated enzymatically using Ascaris succinoxidase (Kmetec 1966). Total carbohydrate was determined with anthrone employing glucose as standard (Mokrasch 1954). ATP was assayed spectrophotometrically (Bueding et al. 1967; Scheibel et al. 1968). 1-[QGlucose, I-[YZ]succinate, 1-[r*C]acetate, lltF]lactate, and UDP-[14C]glucosewere purchased from New England Nuclear, (Boston, MA, USA). Other products employed included hexokinase (EC 1.7.1. l), glucose-6-phosphate dehydrogenase (EC 1.1.1.49), phosphoglucomutase (EC 1.7.5. l), and glucose 1,6-diphosphate from Boehringer-Mannheim (Indianapolis, IN, USA), lactate dehydrogenase (EC 1.1.1.27) from Worthington Biochemical Corp. (Freehold, NJ, USA), glycylglycine, glucose 6-phosphate, and Dowex l-X8 from Sigma Chemical Company, (St. Louis, MO, USA). Glycogen was purchased from MCB (Norwood, OH, USA) and Whatman ET3 1 chromatographic paper from Whatman Inc. (Clifton, NJ, USA). We are indebted to Dr. H. P. Striebel of the Ciba-Geigy Corporation, Basel, Switzerland, for the generous supplies of Amoscanate and its vehicle. In the absence of Amoscanate, administration of the vehicle alone to infected rats had neither a chemotherapeutic effect, nor a metabolic effect on the recovered H. diminutu. RESULTS

Amoscanate was administered to rats infected with Hymenolepis diminuta. After increasing time periods, the animals were sacrificed. To determine whether or not

OF AMOSCANATE

57

Amoscanate had any effect upon the metabolic capacity, the recovered adult parasites were incubated in vitro in the presence of glucose and metabolic parameters of the in vitro incubation were examined. The effects of the duration of Amoscanate therapy on the in vitro metabolism of the parasites were determined by analyzing and quantitating the total amount of each metabolic product which accumulated in the parasite plus the incubation medium (Table I). Up to 8 hr, the longer the Amoscanate therapy prior to sacrifice, the less glucose was utilized by the recovered H. diminuta, and the less succinate and lactate accumulated. The carbohydrate content per milligram of the cestode protein also decreased in response to amoscanate therapy. Of particular importance was the finding of effects after as little as 2 hr of treatment, with quite significant effects after 3 hr of therapy. Similar effects were noted with cestodes which were washed, frozen, and analyzed immediately upon isolation from the host, without further in vitro incubation. In this case, however, the amounts of products isolated were low, since there was no incubation medium to include and only metabolites accumulating inside of the parasite were analyzed. As would be expected, increasing doses of Amoscanate administered to the infected rats also increased the effects on the metabolism of the recovered H. diminuta adults (Table II). Although significant effects were noted at a dose of 75 mg/kg, the highest dose employed (100 mgikg) elicited the most dramatic inhibition of metabolite formation. At this dose, glucose disappearance and succinate accumulation declined approximately 25 and 61%, respectively. ATP content decreased 57% when compared with H. diminuta obtained from untreated rats. Lactate, on the other hand, remained essentially constant in this particular experiment. In other experiments, however, lactate accumulation varied considerably with each preparation in regard to both amounts formed and the effect of Amoscanate, al-

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TABLE I Effects of the Duration of Amoscanate Therapy Prior to the Sacrifice of Infected Rats on the in Vitro Metabolism of the Recovered Hymenolepis diminuta

Hours postAmoscanate therapy 0 2 3 4 6 8

Concentration (pmol/mg protein) Glucose disappearance 3.42 3.19 3.09 2.96 2.88 2.61

% change/8 hr

f f f 5 k k

0.25 0.17 0.100 O.Ob O.l@ 0.W

Succinate formed 0.66 0.57 0.56 0.52 0.44 0.26

-24

* 0.06 * 0.04 +- 0.W k 0.02 t 0.050 k 0.040

Lactate formed 3.05 ?z 0.24 2.70 2 0.23 2.33 f 0.2@ 2.25 f 0.3@ 1.61 ? O.O& 1.43 5 O.O@

-61

mg Total carbohydrate/ mg worm protein 0.64 0.46 0.45 0.41 0.40 0.39

-53

f k * * * ”

0.06 0.07 0.050 0.09 0.030 0.060

-39

Note. 100 mg/kg, single dose of Amoscanate by gastric intubation to infected rats. Worms recovered from rats at indicated time intervals were incubated in the presence of glucose for 90 min at 38 C and quantitation of products were performed as described under Materials and Methods. Rats were treated 18 to 60 days postinfection. Values represent means ? SD; n = eight incubations utilizing helminths from four different rats. * P < 0.05, by Student’s t test in comparison with helminths obtained from rats which were not treated with Amoscanate (0-hr worms).

though most often there was a decline after Amoscanate therapy. It also is worthy of note that parasites which recovered after 8 hr treatment of the host rat with Amoscanate exhibited a motility which was noticeably less than that of the control worms. The effects of Amoscanate on the endogenous carbohydrate metabolism were examined by incubating control and drug-

treated parasites for 90 min in Krebs-Ringer bicarbonate without added glucose (Table III). Once again, the succinate recovered and the ATP content declined considerably. Similarly, as a consequence of Amoscanate therapy the total worm carbohydrate decreased markedly relative to worm protein. These results were consistent with those obtained from incubations in the presence

TABLE II Effects of Increased Doses of Amoscanate to Infected Rats on the Metabolites Accumulated by the Recovered Hymenolepis diminuta After in Vitro Incubation

Amoscanate dosage b&W 0 25 75 100

Concentration (pmol/mg protein) Glucose disappearance 2.76 2.88 2.22 2.04

+ + t k

0.14 0.18 0.130 0.18

Succinate formed 0.97 2 1.07 ” 0.58 2 0.38 ‘-

0.11 0.19 0.18 O.l@

Lactate formed 0.45 0.53 0.67 0.50

+- 0.09 2 0.15 2 0.18 k 0.16

ATP content 0.023 0.023 0.011 0.010

2 0.004 -+ 0.002 2 0.0030 * 0.001~

Note. Amoscanate was administered as a single dose at the specified concentrations via gastric intubation, 8 hr prior to sacritice. All rats were treated 14 days postinfection. Recovered H. diminuta were incubated 90 min in vitro in the presence of glucose as described under Materials and Methods. Values represent means ? SD; n = 4. QP < 0.05, by Student’s t test, in comparison with incubations which did not contain Amoscanate.

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EFFECTS OF AMOSCANATE

TABLE III In Vitro Incubation in the Absence of Exogenous Glucose of Hymenolepis

diminuta Isolated from

Amoscanate-Treated and Untreated Rats Product (pmol/mg protein) Expt

No Amoscanate

8 hr postAmoscanate

Percentage change

Succinate ATP Carbohydrate/ protein0

0.408 k 0.040 0.015 f 0.003

0.224 e 0.041 0.004 ? 0.001

-45 -13

3.31 f 0.089

2.09 f 0.211

-37

Succinate ATP Carbohydrate/ protein0

0.353 f 0.010 0.020 f 0.002

0.244 2 0.008 0.011 * 0.001

-31 -45

4.89 k 0.128

3.31 f 0.067

-32

Component

Note. Amoscanate administered at 100mg/kg, single dose via gastric intubation. Other conditions as described under Materials and Methods, except that glucose was omitted from incubations. Rats were treated 35 and 21 days postinfection in experiments I and II, respectively. Values represent means f SD for duplicate determinations. 0 Carbohydrate/protein is expressed as micromoles of glucose equivalents per milligram protein.

of glucose, and indicate a direct effect upon the utilization of carbohydrate stores within the worm. To determine whether Amoscanate affected glucose incorporation into glycogen and its metabolites, infected rats were treated with Amoscanate and 8 hr posttreatment the cestodes were recovered and incubated for 90 min in the presence of l-

[14C]glucose (Table IV). The levels of isotope found in all three products of carbohydrate metabolism, acetate, lactate, and succinate, declined in the drug-treated worms, with succinate again showing the highest percentage decrease. In addition, the incorporation of labeled glucose into glycogen was reduced by 70%, and the glucose disappearance from the medium decreased

TABLE IV Effects of Amoscanate on the Incorporation of l-[14C]Glucose into Hymenolepis l+C]Glucose Component Acetate Lactate Succinate Glycogen Glucose disappearance Percentage recovery

No Amoscanate 0.346 f 1.037 f 0.588 f 0.671 2 2.956 k

0.072 0.109 0.041 0.114 0.197

89.4

diminuta

incorporated 8 hr postAmoscanate 0.271 0.726 0.213 0.204 2.365

+ 0.062 f 0.082 -c 0.019~ f 0.019 ? 0.124a

Percentage change -22 -30 -64 -10 -20

59.8

Note. Amoscanate was administered at 100 mg/kg as a single dose via gastric intubation 8 hr prior to sacrifice. Rats were treated 14 days postinfection. Except where percentages are listed, all figures represent the mean micromoles of 1C incorporated per milligram worm protein f SD; n = 4. Incubations and isolation procedures were carried out as described in the text. a P < 0.05 by Student’s t test, for corresponding incubations without Amoscanate.

NELSON AND SAZ TABLE V Effect of Amoscanate on Glycogen Synthase I Activity of Hymenolepis diminuta

Expt

Treatment prior to assay None0 None None Incubated* None Incubated None Incubated None Incubated

[*E]Glycogen isolated (pmol/mg protein) No Amoscanate 0.698 0.692 0.461 0.324 0.735 0.878 0.433 0.624 0.421 0.558

k r k 2 f f 2 k k k

8 hr postAmoscanate

0.046 0.092 0.005 0.004 0.027 0.030 0.045 0.023 0.022 0.024

0.537 0.267 0.193 0.327 0.588 0.875 0.362 0.623 0.311 0.543

* f k k 5 f f f 2 k

0.028 0.039 0.027 0.022 0.012 0.003 0.053 0.025 0.066 0.024

Percentage change -23 -61 -58 0 -20 0 -16 0 -26 0

Note. Cestodes were isolated and assayed for glycogen synthase I activity as described under Materials and Methods. Amoscanate was administered to infected rats at 100 mg/kg in a single dose via gastric intubation. All worms were 14 days postinfection prior to therapy, except in experiment 1 where parasites were 90 days postinfection. Values represent mean f SD of duplicate determinations. 0 Worms with no treatment prior to assay were homogenized immediately upon isolation from the host. * Worms incubated in vitro in Krebs-Ringer bicarbonate buffer plus 17 mM glucose for 90 min prior to homogenizing.

20%. These findings indicate an inhibitory effect of Amoscanate on the uptake as well as the utilization of glucose. It should be noted, however, that the recovery of isotope was only 60% from Amoscanatetreated worms and 8% from control worms, again suggesting the accumulation of one or more additional compounds. Amoscanate had no effect on the carbohydrate metabolism when worms were exposed to the drug in vitro. Freshly isolated, nontreated adult worms were incubated for 90 min in Krebs-Ringer bicarbonate with 17 mM Amoscanate. The presence of the drug in the incubation medium had no effect on the disappearance of glucose or the accumulation of succinate or lactate. This was true even when the drug was first suspended in 5% sodium taurocholate in an attempt to increase solubilization and penetration into the parasites. As a consequence of the decreased levels of glycogen and the decreased incorporation of 1-[14C]glucose into glycogen in re-

sponse to Amoscanate therapy, glycogen synthase I was isolated and assayed from drug-treated and control H. diminuta (Table V). When the worms were homogenized immediately upon removal from the host and assayed for glycogen synthase I activity, Amoscanate-treated preparations were inhibited from 16 to 61%. However, if the worms were incubated in vitro for 90 min in the presence of glucose prior to homogenization, the enzymatic activities from drug-treated and control preparations were the same, indicating that the effect of Amoscanate on glycogen synthase I was reversible. To ascertain whether Amoscanate directly inhibited glycogen synthase I in vitro, the drug was added exogenously to the enzymatic assay system. No inhibition was observed under these circumstances. To further elucidate the effect of Amoscanate on glycogen metabolism in H. dimin&a, glycogen phosphorylase activity was examined. AMP independent phosphorylase a activity was approximately 2% lower

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EFFECTS OF AMOSCANATE

TABLE VI Comparison of Phosphorylase Activities in Hymenolepis diminuta from Amoscanate-Treated and Untreated Rats Glucose l-phosphate formed No Amoscanate +3mMAMP

8 hr post-Amoscanate No AMP

Expt

Treatment

No AMP

1

No preincubation Preincubated 10 min at 30 C

10.56 f 1.47 2.56 e 0.28

No preincubation Preincubated 15 min at 30 C Preincubated 15 min, then ATP and Mg2f added

11.65 f 0.28 2.30 + 0.42

15.27 -+ 0.34a 4.77 k O.l&

17.24 2 0.25

17.13 ? 0.47

2

5.19 2 0.58

14.97 + 1.2@ 6.64 2 0.W

+ 3mMAMP 8.05 2 0.31,~

Note. Amoscanate was administered at 100 mg/kg, single dose via gastric intubation. In experiments 1 and 2, worms were 87 and 14 days old respectively prior to Amoscanate therapy. Glucose l-phosphate formed is expressed as nmoles/min/mg protein. The worms were isolated, homogenized, and assayed for phosphorylase activity as described under Materials and Methods. Values represent means f SD; n = 4. Worms were homogenized immediately upon removal from the host and assayed with or without prior preincubation of the homogenates as indicated. Where indicated, preparations were incubated an additional 20 min at 30 C in the presence of 2 mM ATP and 4 mM MgCll prior to assay. a P < 0.05 by Student’s r test, for corresponding incubations without Amoscanate.

in worms isolated from control preparations than from drug-treated preparations (Table VI). Thus, Amoscanate appeared to inhibit glycogen synthase activity, but it stimulated glycogen phosphorylase activity. Preincubation of the extracts at 30 C, which would be expected to allow for the conversion of active phorphorylase a into inactive phosphorylase b, resulted in a decline of 78 and 80% for control preparations (Table VI), but activity in drug-treated preparations, by comparison, decreased only 60 and 69%. Most interesting was the finding that the addition of ATP and MgC& to preincubated samples and subsequent incubation resulted in a complete reactivation of the phosphorylase activity in both the Amoscanate-treated and untreated preparations. Therefore, although Amoscanate therapy stimulated phosphorylase activity, the phosphorylase kinase systems appeared not to be affected by the therapy. To determine whether the effects of

Amoscanate on phosphorylase a were reversible as they were in the case of glycogen synthase, worms were incubated for 90 min in Krebs-Ringer bicarbonate with added glucose prior to homogenization and assay (Table VII). In all instances, glycogen phosphorylase activities were qualitatively similar to those found when the worms were not incubated prior to homogenization and assay. These findings indicate that, in contrast with glycogen synthase activity, the effect of Amoscanate on glycogen phosphorylase activity was not readily reversible. In view of the findings of Voge and Bueding (1980) that ultrastructural changes occurred in schistosomes in response to Amoscanate, ultrastructural studies of H. diminuta were undertaken. The surface topography of the proglottid segments as viewed under SEM appeared to be typical of that described by Ubelaker et al. (1973) for H. diminuta. The surface tegument ap-

NELSON AND SAZ TABLE VII Effects of Amoscanate Therapy on Phosphorylase Activities in Homogenates from Preincubated Intact Hymenolepis diminuta Glucose l-phosphate formed Treatment

No Amoscanate

8 hr post-Amoscanate

No preincubation Preincubated 15 min at 30 C Preincubated, then ATP and MgCl, added

12.73 2 0.18

16.93 + 0.350

2.56 +- 0.51

6.07 k 0.88

19.56 ” 0.75

19.01 f 0.60

Note. Conditions were the same as in Table VI, Expt 2, except that the isolated whole worms (14 days postinfection) were incubated in vitro in Krebs-Ringer bicarbonate buffer plus 17 mM glucose for 90 min at 38 C under 95% N,/5% CO, prior to homogenization and assay. Assays were performed in the absence of added AMP. Glucose l-phosphate formed is expressed as nmoles/min/mg protein. Values represent means f SD; n = 4. * P < 0.05, by Student’s t test, for corresponding incubations without Amoscanate.

peared to be covered with a uniformly dense layer of microtriches. All of the specimens examined, including those exposed to Amoscanate up to 8 hr appeared normal, even under high magnification. Thus, no alterations in surface topography as a result of drug exposure were visible by SEM. The ultrastructure of the outer body wall, including the tegument, and the nephridial system in control specimens was as previously reported (Chatfield and Yeary 1979; Lumsden and Specian 1980; Becker et al. 1981). Sections from untreated H. diminuta body wall and nephridial canal are shown in Figs. 1 and 2, respectively. Three hours treatment of the host with amoscanate still did not reveal abnormalities in the ultrastructure of the parasites (Figs. 3A, B). However, the onset of drug effects became apparent after 4 hr. Blebs started to appear on the external surface of the tegument (Fig. 4A), and a marked decrease in microvilli was noted on the epithelium of the nephridial system (Fig. 4B). Damage was more pronounced 8 hr after Amoscanate treatment of the rat with numerous blebs appearing at frequent intervals on the external surface of the tegument (Fig. SA). In addition, areas of erosion with loss of microtriches were visible on the tegumental surface (Fig. 5B),

and considerable reduction in numbers of microvilli with irregularities in the basement membrane were noted in sections from nephridial canals (Fig. 5C) after 8 hr. DISCUSSION

Chemotherapeutically, Amoscanate is very effective in expelling adult Hymenolepis diminuta from the intestines of infected rats within 24 to 36 hr postadministration of the drug, regardless of the age of the infection. Similarly, regardless of the age of the adult parasites, Amoscanate was found to have remarkable effects on the carbohydrate energy metabolism of H. diminuta. In all experiments, glucose uptake, succinate accumulation, ATP levels, and total carbohydrate per milligram of worm protein decreased in response to therapy of the infected rat with Amoscanate. In most experiments lactate accumulation also was depressed, but not consistently. Not only was glucose uptake depressed in response to therapy, but also the incorporation of l[14C]glucose into worm glycogen and its metabolites was depressed, suggesting the possibility of a lesion in transport. The depression of glycogen levels in the parasite exposed to drug therapy also might be a consequence of the observed inhibition of

Hymenolepis

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EFFECTS OFAMOSCANATE

FIG. 1. (A) Tegumental section of Hymenolepis diminuta untreated control, showing tegument with microtriches on surface and subtegumental cells. x9240. Bar = 1 km. (B) Tegumental section at higher magnification of H. diminura untreated control, showing microtrich layer, tegument, and subtegumental cells. x 15,400. Bar = 1 pm.

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NELSON AND SAZ

FIG. 2. (A) Section of nephridial canal of Hymenolepis diminufa untreated control, showing microvilli. x9240. Bar = 1 km. (B) Section of nephridial canal at higher magnification of H. diminuta untreated control, showing basement membrane and microvilli. x 22,100. Bar = 1 pm.

Hymenolepis diminuta: EFFECTS OFAMOSCANATE

FIG. 3. (A) Section of tegument of Hymenolepis diminuta 3 hr. post-treatment, 100 mg/kg, showing no abnormalities. x 15,400. Bar = 1 pm. (B) Section of nephridial canals of H. diminuta 3 hr posttreatment, 100 mg/kg, appearing undamaged. ~26,180. Bar = 1 pm.

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FIG. 4. (A) Section of tegument of Hymenolepis diminuta 4 hr post-treatment, 100 mg/kg, showing blebs on surface. ~22,100. Bar = 1 pm. (B) Section of nephridial canal of H. diminuta 4 hr posttreatment, 100 mg/kg, showing marked decrease in numbers of microvilli present on surface epithelium. x9240. Bar = 1 pm.

Hymenolepis

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EFFECTS OF AMOSCANATE

FIG. 5. (A) Section of tegument of Hymenolepis diminuta 8 hr post-treatment, 100 mg-kg, showing several blebs on surface of tegument. x 18,480. Bar = 1 pm. (B) Section of tegument and subtegument of Hymenolepis diminuta 8 hr post-treatment, 100 mg/kg, showing several areas of erosion or disruption of microtriches. x6160. Bar = 1 pm. (C) Section of nephridial canal of H. diminuta 8 hr posttreatment 100 mg/kg, showing considerable reduction in numbers of microvilli with irregularities in the basement membrane (arrow). x 9240. Bar = 1 pm.

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glycogen synthase I and the stimulation of glycogen phosphorylase. It seems unlikely that the findings described with H. diminuta could explain the chemotherapeutic effect of Amoscanate on either schistosomes or tilariids, since the latter helminths are not destroyed for several months after therapy of the rodent host (Bueding et al. 1976; Saz et al. 1977). However, Voge and Bueding (1980) reported ultrastructural changes on the schistosome surface associated with Amoscanate therapy. These changes appeared rapidly after administration of the anthelmintic. Similarly, ultrastructural changes of the H. diminuta surface were noted shortly after Amoscanate therapy of the infected rat host, again suggesting an alteration in transport. In addition to bleb formation and eventual erosion at the surface of H. diminuta, there was a disappearance of microvilli from the nephridial canals. However, the alterations in ultrastructure were not observed until 4 hr post-Amoscanate therapy. In contrast biochemical effects were apparent after only 2 hr post-Amoscanate therapy. These tindings suggest that the changes in ultrastructure may be secondary to the biochemical lesions. Amoscanate affects a number of biochemical sites in the parasites, as might be expected of an isothiocyanate derivative. Which one or more of these sites is responsible for the chemotherapeutic effect has not been established. The effects of the anthelmintic on the uptake and energy metabolism of Hymenolepis diminuta, however, might play a major role in the expulsion of the parasites from the host intestine. ACKNOWLEDGMENTS The authors wish to thank Ms. Carol A. Strenkoski and Dr. Lloyd A. Davidson for their helpful suggestions and technical assistance. This investigation was supported in part by Grants AI-09483 and AI-07030 from the U.S. National Institutes of Health.

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