The effects of dl -tetramisole and rafoxanide on tricarboxylic acid cycle enzymes of Haemonchus contortus, in vitro

Veterinary Parasitology, 13 (1983) 3 3 3 - 3 4 0 Elsevier Science Publishers B.V., Amsterdam - - Printed in The Netherlands

333

THE EFFECTS OF D L-TETRAMISOLE AND RAFOXANIDE ON TRICARBOXYLIC ACID CYCLE ENZYMES OF HAEMONCHUS CONTORTUS, IN VITRO

RANBIR KAUR and M.L. SOOD*

Department of Zoology, Punjab Agricultural University, Ludhiana-141 004 (India) (Accepted for publication 28 April 1983)

ABSTRACT Kaur, R. and Sood, M.L., 1983. The effects of DL-tetramisole and rafoxanide on tricarboxylic acid cycle enzymes of Haemonchus contortus, in vitro. Vet. Parasitol., 13: 333--340. Various enzymes of the tricarboxylic acid cycle (TCA) viz., aconitase (E.C. 4.2.1.3), isocitrate dehydrogenase (E.C. 1.1.1.42), succinate dehydrognease (E.C. 1.3.99.1), fumarate reductase (NADH: fumarate oxido-reductase), fumarase (E.C. 4.2.1.2) and maltate dehydrogenase (E.C. 1.1.1.37) were detected in adult Haemonchus contortus (Nematoda: Trichostrongylidae), in vitro. Low activities of aconitase and isocitrate dehydrogenase suggested that the TCA cycle has a minor function and the pathway of CO2 fixation is the major pathway in the energy metabolism of the parasite. In vitro incubation in Tyrode's solution had no significant effect on TCA cycle enzymes and the worm was able to maintain normal metabolism for 12 h. The effects of DL-tetramisole and rafoxanide on various enzymes of the TCA cycle were studied in adult H. contortus. At 50 ~g m1-1 varying degrees of inhibition of succinate dehydrogenase and fumarate reductase activities were observed. At the same concentration, the activities of other enzymes remained unaltered.

INTRODUCTION Parasitic helminths found in environments with low oxygen tension possess a predominantly anaerobic energy metabolism, despite the fact that they may take up measurable amounts of oxygen from an anaerobic environment in vitro (KShler and Stahel, 1972; Ward, 1974). Earlier views of Rogers (1948) suggest that Haemonchus contortus (Rud., 1803), the most pathogenic nematode parasite of sheep, goats and other ruminants, utilizes oxygen even at low partial pressures. Recently, Ward and Huskisson (1978) have reported that in the adult, both aerobic and anaerobic metabolisms could be essential and occurred simultaneously. The presence of the *Author to whom correspondence should be addressed.

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334 tricarboxylic acid (TCA) cycle in larval H. contortus has already been investigated (Ward and Schofield, 1967). Therefore, the first objective of the present investigation was to study in adult H. contortus various enzymes of the TCA cycle, under in vitro conditions. The increase in the knowledge of metabolism of parasitic helminths has shown that in the TCA cycle, the succinate dehydrogenase--fumarate reductase (SDH--FR) system functions as a respiratory chain, and appears to be a particularly vulnerable point for chemotherapeutic interference. In nematodes, this system has received much attention as a possible site of action of the anthelmintics cambendazole, thiabendazole, levamisole and mebendazole (van den Bossche and Janssen, 1967, 1969; van den Bossche, 1972; Prichard, 1970, 1973; Malkin and Camacho, 1972; Romanawski et al., 1975; Boczon, 1976; K5hler and Bachmann, 1978). Thus, the second objective of the present study was to investigate the effects of two anthelmintics; D L-tetramisole (TMS) (Imperial Chemical Industries, Madras, India), nilverm and rafoxanide (RFX) {Merck, Sharp and Dohme, Bombay, India), ranide. In carbohydrate metabolism of H. contortus, of these two drugs, TMS has so far only been shown to affect fumarate reductase (Prichard, 1973). The effect of RFX has not y e t been studied on the carbohydrate metabolism of any nematode. Therefore, a comparative effect of these two drugs at one concentration has been studied on various enzymes of the TCA cycle, with special reference to SDH--FR system. MATERIALS AND METHODS Living adult specimens of H. contortus collected from the abomasa of infected goats (Capra hircus) were used for the present study. Worms were washed thoroughly in 0.9% saline and freed from the adhering host material. In vitro anthelmintic studies

For in vitro anthelmintic studies, various concentrations of TMS ranging from l 0 to 50 pg ml 1 were added to sterile conical flasks, each containing 15 worms in 25 ml of Tyrode's solution (Ward, 1974). These flasks were incubated at 37°C in air-CO2 (95/5). However, 100% mortality was achieved only with the highest concentration (50 pg m1-1 ), at 10 h. This concentration was, therefore, selected for subsequent studies. For comparative studies, the same concentration (50 pg m1-1 ) of rafoxanide was used and 100% mortality was achieved at 14 h. The worms were, therefore, fixed before the time required to kill them had elapsed, i.e., at 8 h with TMS and 12 h with RFX treatments.

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Preparation o f enzyme extracts The pooled worms of fresh (non-incubated), control (Tyrode-incubated) and drug (TMS and RFX) treated groups were homogenized in ice-cold 0.25 M sucrose in a teflon pestle homogenizer (Remi Udyog, Bombay) to give 10% (w/v) extracts and then sonicated in an ultrasonicator (model VPL-P2, Vibronic Pvt. Ltd., Bombay, India). The homogenate was then centrifuged at 700 g at 0°C for 10 min in a K24 centrifuge (Jenetzki, GDR). The supernatants were decanted and the pellets were resuspended in 0.25 M sucrose and recentrifuged. The supernatant obtained was decanted and pooled with the first supernatant. These pooled supernatants were centrifuged at 10 000 g for 10 min. The sedimented mitochondrial pellet was resuspended in 0.25 M sucrose and recentrifuged. The pellet represented the mitochondrial fraction, and was used as a source of various enzymes. Proteins were determined by the m e t h o d of Lowry et al. (1951). Enzyme activities were expressed as t~mol substrate f o r m e d / m i n / m g protein.

Enzyme assays Aconitase (AC) activity was determined according to the procedure described by Anfinsen (1955). The 3-ml assay medium consisted of 2.9 ml of citrate (0.003 M)-buffer (0.005 M) solution (pH 7.4) and 0.1 ml of the homogenate. The decreased optical density (O.D.) associated with NADH oxidation was recorded at 240 nm at intervals of 15 s for a few mins at 25°C. Isocitrate dehydrogenase (ICDH) activity was measured by following increase in O.D. associated with NADP reduction at 340 nm according to the m e t h o d of Ochoa (1955). The components of the reaction mixture consisting of 0.3 ml glycylglycine buffer (2.5 M, pH 7.4), 0.1 ml MnC12 (0.018 M), 0.1 ml NADP (0.00135 M), 0.1 ml of isocitrate (0.003 M), water and 0.01 ml of the homogenate were added to a 3 ml cuvette. Readings were taken against a blank containing all the components except NADP at intervals of 15 s for 1 or 2 min. Succinate dehydrogenase (SDH) activity was measured according to the m e t h o d described by Arrigoni and Singer (1962). The mitochondrial preparations were preincubated with succinate for activation of SDH activity. For this, 0.1 ml of mitochondrial preparation, 0.1 ml of potassium phosphate buffer (0.5 M, pH 7.6), 0.05 ml succinate (0.5 M), 0.25 ml water were incubated at 37°C for 7 min. For each assay 0.5 ml of the preincubated mitochondrial preparation was added to 0.1 ml of 2% phenazine methosulphate and 2.9 ml of Cocktail solution. Optical density at 600 nm associated with NADH oxidation was measured against water at 30-s intervals for 2 min. Fumarate reductase (FR) activity was determined according to the method described by Prichard (1970). The decreased O.D. associated with NADH

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oxidation was recorded at 340 nm in the reaction mixture contained in 3.0 ml of 1 mmol of potassium phosphate buffer (0.4 M, pH 7.0), 10 ~mol MgC12, 0.3 pmol NADH, 10 pmol sodium fumarate, water and 0.1 ml of the homogenate. All reactions were maintained at 37°C, except the protein fraction which was kept at 0oc. Components through NADH were mixed and incubated for 2 min at 37°C before fumarate was added to initiate the reaction. Fumarase (FM) activity was determined by recording the decrease in O.D. associated with NADH oxidation according to the m e t h o d described by Massey (1955). The constituents of the FM assay consisted of 2.5 ml of potassium phosphate buffer (0.033 M, pH 7.3), 0.2 ml of sodium fumarate (0.017 M), water and 0.1 ml of homogenate in a total volume of 3 ml mixed in a quartz cell having 1 cm light path. NADH oxidation was recorded at 340 nm. Malate dehydrogenase (MDH) activity was determined according to the procedure described by Englard and Siegel (1969). The reaction mixture contained 2.5 ml of glycine--NaOH buffer (0.12 M, pH 10.0), 0.3 ml of L-malate (0.85 M) 0.2 ml of NAD + (37.5 mM) and 0.01 ml of the homogenate. The rate of NADH formation was conveniently measured spectrophotometrically by the increase in O.D. at 340 nm. RESULTS

The relative activities of the TCA cycle enzymes from the mitochondrial fraction of H . c o n t o r t u s a r e summarized in Table I. The present studies showTABLE I E f f e c t s o f D L - t e t r a m i s o l e ( T M S ) a n d r a f o x a n i d e ( R F X ) o n T C A c y c l e e n z y m e s in H a e m o n c h u s t o r t u s , in v i t r o Enzyme unit # m o l m i n -l m g -I protein

Nucleotide measured

Aeonitase

NADH oxidation

0.0034

Isocitrate dehydrogenase

NADP reduction

0.025 -+0.0023

0.027 -+0.004

+ 7.4(N8)

0.030 -+0.004

+16.6(NS)

Succinate dehydrogenase

NADH oxidation

0,184 -+0.0006

0,098 -+0.001

--46.7(S)

0.106 -+0.001

--42.3(S)

Fumarate reductase

NADH oxidation

0.366 -+0.028

0.126 -+0.05

--65.5(S)

0.300 -+0.05

--18.0(S)

Fumarase

NADH oxidation

1.23 -+0.046

1.26 -+0.08

+2.3(NS) 1.26

1.20 -+0.011

1.23 +-0.02

+ 2.4(NS)

Malate dehydrogenase

NAD reduction

Control (Tyrodeincubated)

TMS treated

0.0036

% Change RFX treated

% Change

+5.5(NS)

+10.5(N8)

0.0038

con-

+ 2.3(NS)

-+0.08 1.24 -+0.02

+ 3.2(NS)

V a l u e s are e x p r e s s e d as m e a n -+ S.E. ( n = 4 ) . S = s i g n i f i c a n t l y d i f f e r e n t f r o m c o n t r o l a t P < 0 . 0 5 ; NS = non-significant.

337 ed the presence of most of the enzymes of the TCA cycle, although citrate synthase and ~-ketoglutarate dehydrogenase were n o t assayed. In vitro incubation results

Activities of various enzymes in control I (control of TMS treatment) and control II (control of R F X treatment) did not differ significantly from those in the non-incubated worms, although a non-significant increase was observed. Therefore, a mean control for each enzyme has been shown in Table I. Effects o f anthelmintics on the e n z y m e activities

The effects of TMS and R F X on the rate of NADH oxidation in AC, SDH, FR, FM-catalyzed reactions, NADP reduction in ICDH-catalyzed reaction and on the rate of NAD reduction in MDH-catalyzed reaction are summarized in Table I. Varying degrees of inhibition of the TCA cycle enzymes were observed with the drugs. Both the drugs inhibited the activities of SDH and FR. However, after TMS treatment, more inhibition of the enzymes was observed than after R F X treatment. The drugs had no significant effect on the activities of AC, ICDH, FM and MDH. After treatment with TMS, SDH and FR activities were reduced to 46.7% and 42.3%, respectively, and after R F X treatment to 65.5% and 18.0%, respectively. The present results indicated that in the TCA cycle, the c o m m o n site of action is the S D H - - F R pathway. SDH activity was more sensitive to both the drugs as judged by its maximum inhibition. DISCUSSION The present biochemical studies have shown the presence of most of the enzymes of the TCA cycle in adult H. contortus, though the activities of citrate synthase and a-ketoglutarate dehydrogenase were n o t investigated. However, low activities of AC and ICDH have been observed and suggest that the complete cycle may be of relatively minor importance in the overall metabolism of the parasite. Similar conclusions concerning the importance of the complete cycle have been drawn for H. contortus larvae (Ward and Schofield, 1967) and Ascaris (Barrett, 1976). Besides, the low activities of AC and ICDH, high activities of SDH, FR, FM and MDH were detected in adult H. contortus. Similar observations have been made in Ascaridia galli and Rhabdias bufonis (Srivastava et al., 1970; Anya and Umezurike, 1978). Carbohydrate breakdown in Ascaris lumbricoides involves part of the TCA cycle essentially working in reverse from oxaloacetate (OAA) to succinate, and it seems unlikely that a classical TCA cycle is involved in this nematode (Barrett, 1976). In parasites which fix CO2, the electron

338 transport systems are involved in the reduction of fumarate to succinate. As reported earlier (Ward and Huskisson, 1978, 1980}, H. contortus also fixes CO2 and the low activities of AC and ICDH and high activities of FR, SDH, FM and MDH detected during the present studies, further support the above view. The present data reveal that CO2 fixation is the major pathway and that the TCA cycle has at the most a minor function, possibly concerned with the interconversion of carbon skeletons in aminoacid metabolism (Oya et al., 1962, 1965). In vitro incubation in Tyrode's solution for 12 h had no significant effect on the activities of the TCA cycle enzymes. These results indicated that the worms were able to maintain normal metabolism for the specified time period. Treatment with TMS and RFX had no significant effect on the activities of AC, ICDH, FM and MDH. However, after treatment, activities of SDH and FR were inhibited. Van den Bossche and Janssen (1967) also observed inhibition of SDH activity with TMS in Heterakis, Trichuris, Ascaridia, Chabertia, Bunostomum and Nematodirus. Previous work on Haemonchus contortus has shown that FR activity was inhibited with tetramisole, thiabendazole, cambendazole, mebendazole, morantel tartrate and disophenol (Prichard, 1970, 1973; Malkin and Camacho, 1972). TMS has also been shown to have a marked effect on cholinesterase (Kaur and Sood, 1982). Thus, it is evident that in H. contortus, TMS affects both neuromuscular system and carbohydrate metabolism. Both SDH and FR activities were inhibited by cambendazole, thiabendazole and levamisole in Aspicularis tetraptera and Ascaris suum (Comley and Wright, 1981). Inhibition of the SDH--FR system is extremely important since fumarate is the terminal electron acceptor in the metabolism of a number of helminths (Malkin and Camacho, 1972). The reduction of fumarate to succinate also regenerates NAD ÷ and it appears to produce mitochondrial ATP (Kmetec and Bueding, 1961; Saz, 1971). Therefore, the inhibition of this enzyme system may not only prevent succinate formation, but also the production of ATP, the regeneration of NAD + as well as interference with terminal electron transport. Present studies showed that after treatment with TMS and RFX, the activities of FM and MDH remained unaltered. Similar observations have also been made in A. lumbricoides with DEP (Kunitomo and Nagakura, 1974) and in Onchocerca volvulus with suramin (Walter and Schulz, 1980). It is evident from the data presented that in vitro, the drugs have an inhibitory effect on the SDH--FR system. CONCLUSIONS The present biochemical studies have indicated the presence of most of the enzymes of the TCA cycle in adult H. contortus. Low activities of AC and ICDH suggest that the TCA cycle may n o t be of prime importance in this parasite. In vitro incubation in Tyrode's solution shows that the

339 w o r m is a b l e t o m a i n t a i n n o r m a l m e t a b o l i s m f o r 12 h. T h e i n h i b i t o r y e f f e c t s seen f o l l o w i n g t r e a t m e n t w i t h 50 p g m1-1 o f T M S a n d R F X o n S D H a n d F R a c t i v i t i e s c o u l d be e x p l a i n e d on t h e basis o f d e c r e a s e d A T P synthesis along the SDH--FR system. ACKNOWLEDGEMENTS T h e a u t h o r s t h a n k P r o f e s s o r S.S. G u r a y a , H e a d o f t h e D e p a r t m e n t f o r e n c o u r a g e m e n t s a n d t h e l a b o r a t o r y facilities. R.K. also t h a n k s t h e a u t h o r ities o f C . S . I . R . , N e w D e l h i f o r t h e g r a n t o f J u n i o r / S e n i o r R e s e a r c h F e l l o w ships t o her.

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