biochemical and immunochemical effects of certain ...ir.amu.ac.in/7353/1/ds 2495.pdf · tabel -5...
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BIOCHEMICAL AND IMMUNOCHEMICAL EFFECTS OF CERTAIN ANTHELMINTICS ON
SOME GASTROINTESTINAL HELMINTHS
DISSERTATION SUBMITTED FOR THE DEGREE OF
MUittV of $I|tlQS[QpI)P P A. ®T^. IN
ZOOLOGY
*v,-'
BY
GUbSHAN ZIA
SECTION OF PARASITOLOGY DEPARTMENT OF ZOOLOGY
ALIGARH MUSLIM UNIVERSITY ALIGARH (INDIA)
1994
ii
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I }
DS2495
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$ ?C*>>~v>'
J</c»»*<; Aligarh Muslim University
Section of Parasitology, Department of Zoology A M U . , Aligarh - 202 002 [INDIA]
( J ) . (0571) 401647/400922, Ext, 302
Wajih A. Nizami
Dated A?.:^.:.^"^."^^
This is to certify that the dissertation entitled
"Biochemical and immunochemical effects of certain anthelmintics
on some gastrointestinal helminths" submitted by Ms Gulshan Zia,
embodies original work done by the candidate herself. The entire
work, was carried out under my supervision and that I allow her to
submit the same in partial fulfilment of the reqiilromnntn for the
degree of Master of Philosophy in Zoology of this University.
Prof. W.A. Nitimi
(Supervisor)
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CONTENTS
Page
List of Tables ;
List of Figures 11
List of Plates ijl
AcJcnowledgemenLs JV
CHAPTER I INTRODUCTION ]
CHAPTER II HISTORICAL REVIEW 9
CHAPTER III MATERIALS AND METHODS 31
CHAPTER IV RESULTS ^
CHAPTER V DISCUSSION 55
CHAPTER VI SUMMARY Q 3
BIBLIOGRAPHY 66
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LIST OF TABLES
Table -1 Enrichment of surface plasma membrane enzyme activities in comparison with fresh worm homogenate of G.explanaturn.
Table -2 Enrichment of surface plasam membrane enzyme activities in comparison with fresh worm homogenate of G. crumenifer.
Table -3 Effect of anthelmintics on membrane bound marker enzymes of G. crumenifer ( soluble fraction).
Table -4 Effect of anthelmintics on membrane bound marker enzymes of G. crumenifer ( membrane fraction).
Tabel -5 Effect of anthelmintics on membrane bound marker enzymes of G. explanatum ( soluble fraction).
Table -6 Effect of anthelmintics on membrane bound marker enzymes of G. explanatum. ( membrane fraction).
Table -7 Effect of some anthelmintics on in vitro 3
uptake of -glucose in G. crumenifer.
Table -8 Effect of oxyclozanide on the antibody titer in rabbit.
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48
50
51
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56
63
(0
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LIST OF FIGURES.
Fig 1. Flow diagram for the isolation of surface plasma membrane of amphistomes.
Fig 2. Chemical structures of the anthelmintic drugs used.
Fig 3. Effect of mebendazole on in vitro uptake of 3 H-glucose by G^ crumenifer.
Fig 5, Effect of Rafoxanide on in Yi.tro uptake of 3 H-glucose by G. crumenifer.
C'l)
Page
32
36
57
Fig 4. Effect of oxyclozanide on in vitro uptake of 58 3 H-glucose by G. crumenifer.
59 Fig 6. Diagrammatic representation of precipitation
peaks following reversed rocket immune- co electrophoresis.
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LIST OP PLATES
PEiATE 1. Infected liver and rumen showing Giqantocotyle explanatum and Gaatrothylax crumenifer infections respectively.
PEJATE 2. Whole mount preparations of Gastrothvlax crumenifer and Giqantocotyle explanatum.
PLATE 3. Trasmission electron micrographs of Gastrothylax crumenifer and Giqantocotyle explanatum surface plasma membranes isolated after 20 min of detergent treatment.
PLATE 4. Transmission electron micrographs of surface plasma membranes of Gastrothylax crumenifer showing trilaminate structure, puffed membranes and pores.
Plate 5. Reversed rocJcet Immunoelectrophoresis of control and experimental sera against partially purified membrane antigens of Gastrothylax crumenifer.
Page
7
6
AA
A5
61
(iii)
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ABBREVIATIONS USED
ATP CaCl
2 cm DDW DMSO g h
HBSS KH PO
2 4 M mA MBZ MOS min mm mM N nm nmole OXCL Pi RFZ Sec TCA ugm ul v/v w/v
Adenosine Triphc phate Calcium Chloride
Centimetef Double Diattiled Water Dimetnyi sulfoxide Gram Hour Hank's Balanced Salt Solution Potassium dihydrogen phosphate
Molar Milliampere Mebendazole Membrane disrupting solution Minute Mill meter Milli molar Normal Nano meter Nano mole Oxyclozanide Phosphorus Rafoxanide Second Trichloroacetic acid Micro gram Microliter volume/volume Weight/Volume
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ACKNOWLEDGEMENTS
I fall short of words to express my gratitude to ray-
supervisor Professor W.A. Nizami for his inspiring guidance,
encouragement and keen interest throughout the course of this
study. Infact it was a great previlage to work under his
guidance.
Sincere thanks are due to former chairman Professor M.M.
Agarawal, present chairman Professor A.K. Jafri, department of
Zoology, A.M.U. for providing the necessary laboratory
facilities.
I wish to exress my heartfelt gratitude to my parents for
their love, encouragement and moral support.
Special thanks to my colleagues Dr. S.M.A. Abidi, Dr.Parveen
Khan, Dr. Malik Irshadullah, Mr M. Khalid Saifullah , Mr Afsar
Ali, Mr Gul Ahmad and Ms Nazneen B. Khan for providing help
during the tenure of this work.
I also record my sincere thanks to my friends who never left
me alone with my problems.
I am thankful to Mr Mirza Rais Baig for typing this
manuscript and Mr Kalam & Mr Samiuddin for collecting the
parasites.
Finally I am grateful to A.M.U. Aligarh for providing the
financial assistance.
( Gulshan Zia)
(iv)
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1
INTRODUCTTION
Arophiatomes are thick bodied conical or cylindrical
digeneans, distinguished from other forms by posteriorly located
acetabulum, responsible for firm attachment to the host tissue.
Infection of amphistomes are commonly found in sheep, goats,
cattle and water buffaloes in India as well as in other tropical
countries. The disease amphistomiasis is caused by the massive
infection of immature paramphistomes and this disease is
characterized by acute gastro-enteritis with high morbidity and
mortality rates, particularly in young stock. Rumen parasites
generally render a low pathogenicity causing acute catarrhal and
haemorrhagic inflammation in the abomasum, duodenum, and jejunum
with associated anaemia, hypoproteinaemia. While the amphistomes
parasitizing the bile duct cause haemorrhage, pronounced
periductal fibrosis and other hyperplastic changes (Kulasiri and
Seneviratne, 1956; Arora and Kalra, 1971; Jha et al., 1977),
Migrating immature paramphistomes in intestine cause severe
pathological changes characterized by general weakness,
increasing anorexia and polydypsia.
In a survey from our laboratory on the epidemiology of
amphistomiasis 71.4* of buffaloe's rumen were found infected with
different species of amphistomes with varying intensities of
Gastro' yla; crumenifer (32.7%), Fischoederius elongatus (6.0*),
Paramphistomum epic1itum (51.9 X), Qrthocoelium scollpcoellum
(16.IX). While in liver, Gigantocotyle explanatum infection was
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observed in 19.6\ animals which was most widespread amphistomes
of bile duct (Mattison et al., 1994).
India is basically an agricultural country, where livestock
contribute considerably and provide 3 2* of the total energy
requirement of rural areas (Odend'hal, 1972). Acoording to FAO
(1986) production report asian countries have 96* of the world's
buffalo population, of which India has the largest population
(54\) of buffaloes. In 1985, total yield of milk and meat from
India was 21.391 and 0.139 million tonnes, while 32 and 1.0
million tonnes respectively were produced by the remaining world.
Besides dairy products, buffaloes form the main sources for
various agro based industries like milk, meat leather industries,
transport as well as serve as a source of fuel and fertilizers.
It has been estimated that the animal husbandry generated
1,08,640 million in 1984-85 which is roughly 18* of the total
agricultural out put. The dairy products are an important source
of nourishment to our diet. Buffaloes contributes 58* of the
milk though they form only 30* of the total bovine (Cattle and
buffaloes) population (Acharya, 1988).
Despite their importance in the country's economy the net
contribution made by animal wealth is not very satisfactory. Poor
animal health has been considered as one of the main reason for
meagre output. Besides viral, bacterial and protozoan infections,
a bewildering array of helminth parasite cause heavy morbidity
and also mortality in livestock during epidemic out break. In
view of losses incurred by such parasitic iniections, it is
esrsential that we should protect our livestock from these
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infections. It has been suggested that the milk and meat
production and draught animal power can be imporved through
selective breeding and effective animal management. However,
desired results can only be obtained if equal attention is paid
to the health of animals.
In order to control any parasitic infection it is essential
to have a complete background of parasite metabolism. Most of
the physiological information on the adult trematodes has come
from observation on the functional morphology of the various
organ system like tegument, sucker, alimentary canal,
parenechyma, excretory system, reproductive system etc.
The tegument of trematodes consists of an outer anucleated
syncytial layer which is contributed by the inner nucleated
layer comprising the tegumental cells and other cellular
organelles. It is externally covered by plasma membrane which is
involved in many physiological processes including the absorption
of nutrients, excretory processes and immunological interactions.
In addition to this, surface plasma membranes are also involved
in protection from host digestive enzymes, immunoglobulin attack,
and transport of a large number of solutes by a variety of
absorption mechanisms (Diffusion, active transport, pinocytosis
etc). The tegument of helminths is known to be covered with an
unstirred layer which includes the glycocalyx. It is largely
composed of glycoprotein, with projecting side chains of
oligosaccharides and gangliosides, uoth bearing terminal sialic
and other acid residues. The various ionic components of host
origin are absorbed to the glycocalyx by electrostatic
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t
interaction. Further, the glycocalyx may act as "cation exchange
reain" by concentrating those cations which are necessary to
maintain maximum enzyme activity (Lumsden, 1972).
Therefore, the tegumental surface of the trematodes form an
interface vMith the habitat and it is site for a variety of
biochemical processes like absorption, digestion, "protection"
and "infromation transfer". Each of these processes play a vital
role in maintaining the parasitic mode of life of these
organisms. Knowledge of the surface plasma membranes is therefore
fundamental to understand the host- parasite relationship, which
provides basic information for the development of any effective
control programme through immunological or pharmacological means.
Those drugs that cause dysfunction at the surface should be
considered highly sucessesful therapeutic agents.
Anthelmintic treatment for the control of immature and
immature paramphistomiasis has been practised at various levels.
The removal of adult infections is probably of little direct
benefits to the animal but may be prophylactic, as it serves to
reduce the reservoir infection for intermediate host. Several
anthelmintics have been used against immature stages in sheep and
goat and occassionally proved effective in the field. However, no
drug is available showing high efficacy against adult flukes and
the availability of highly effective and safe drugs for the
treatment of immature worms are very limited. Various drugs have
been tested against these infections by a number of workers
(Ahmad, 1984 and Boray, 1986) and suggested that the mode of
action of anthelmintics is generally associated with energy
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yielding processes, nutrient upta)ce mechanism, and neuromuscular
coordination.
Although, considerable progress has been made in recent
years on the chemotherapeutic effect of various anthelmintics and
their mode of action but very little is known about their
influence on specific immune status of the treated host. Such
information will generate indirect evidence on effectiveness of
anthelmintics.
As mentioned earlier, the control of parasitic diseases
relies largely on prophylactic or therapeutic application of
antiparasitic drugs. In many instances these measures are only
partially effective and lead to the selection of drug resistance
within the parasite population. Therefore chemotherapy required a
rational approach rather than emperical approach. Besides this,
drugs have appreciable with holding capacity in biological system
and produce profound toxic effects which is an important aspect
and can not be ignored.
Available literature on this aspect clearly reveal that
anthelmintic drugs affect the immune system of the host. It has
been reported that antiparasitic compounds affect the lymphoid
organs, T and B cells, lymphocyte count, as well as bone marrow
progenitor cells. Besides this, the antibody titer is also
affected. (Von Behren et al., 1983 ; Goldstein et al., 1969 and
Thigpen et al., 1975). Such immunomodulatory action of
anthelmintics may result in impairement of host immune system,
leading to increased susceptibility to subsequent infections and
loss of resistance to parasitic infection.
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0
study of the relationship between the immune system and
chemotherapy in parasitic diseases is still relatively
unexplored. Many of the anthelmintic agents currently in use are
Icnown to require an intact immune system to eliminate the
parasites from the host. To date, combined immunostimulation and
chemotherapy will only more effective than chemotherapy alone for
helminth parasite treatment.
In view of the above facts, the present study was undertaken
to investigate the molecular action of some Icnown anthelmintics
on the surface plasma membrane bound enzymes. Since the surface
plasma membrene seems to be primary target for the drug action 3
and involve in trasmembranosis, therefore H-glucose uptake was
analysed in presence and absence of drugs. In addition to this,
the effect of oxyclozanide on the antibody titer against the
membrane antigen was also investigated by using reversed rocket
Immunoelectrophoresis.
It is expected that the outcome of this study will
definitely contribute in designing the pharmacological control
of these economically important group of parasites. Further, this
study will provide some data relevant to the immunoprotection
studies for an epidemic area of amphistomiasis.
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Plate ]: Infected liver and rumen sho wing a: Heavy infection of liver amphistome Gigantocotyle exElanatum. Bile duct (arrow heads) enlarged and extensive liver damage can be seen. b: Heavy infection of Gastrothylax crumenifer in the anterior dorsal sac of the rumen of buffalo. The flukes often occur in clamps (arrow). Mucosa at the attachment site becomes depigmented.
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Plate 2: Whole mount preparation of a: Gagtrothylax crumenifer t): Gigantocotyle explanatura staintd with alcoholic borax carmine (scale bar 2 urn). Ph: Pharynx, U: Uterus, G: Gut, VP: Ventral pouch, Te; Testes. Ov: ovarv: Ovarv. AC: Acetabulum.
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HISTORICAL REVIEW
Parasites pose a continual and unacceptable threat to the
well being of human being and to the farm animals all over the
world. The cost of parasites in terms of human misery is
incalculable,, although various reports are available on economic
loss from our farm animals. Parasites have evolved ways of
surviving in nutritionally rich, but immunologically hostile
environment of their host. These adaptations that make them
unique and fascinating organisms to study. Parasites generally
display a combination of biological and chemical adaptations
unique in animal world and at the same time display a range of
methods of evadingthe host immune response in order to establish
a delicate and harmonious host- parasite relationship.
In view of the clinical and economic importance of these
parasites, eradication of parasites has been the goal of various
nationnl and international organizations. Control of parasites of
livestock usually aims at reducing the parasitism to a level
having no effect on productivity provided that it is economically
feasible. For this, different methods of control have been
adopted by the parasitologists.Chemotherapeutic and immunological
control have received considerable attention during the recent
past. In the former approach a number of new compounds have been
synthesiser an screened for their efficacy. The available
literature indicates that in most of the cases the mode of action
is not completely known or if partially known then its
toxicological aspects have not been investigated. The main aim of
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10
pharmacologists is to control the parasites either at individual
or mass level. Today, various antiparasitic compounds are
available but the effect of these compounds on the host
particularly at immunological level is more or less neglected.
Parasite chemotherapy has attracted considerable attention
over the past two decades and numerous reports on drug
development, their mode of action and laboratory and field
trials are available. Comparative studies on various drug
formulation, their chemistry and the selective toxicity have also
been investigated. In 1909 Paul Ehrlich laid down the foundation
of chemotherapy of parasites and proposed that the inhibition of
enzymes that were crucial to the parasites but not to the host
might be the basis of a rational approach to the chemotherapy of
parasites.
Von Brand (1973) suggested that most drugs interfere with
the enzyme systems by inhibiting them and thus interfere with the
metabolic processes. Such studies would be of immense value in
knowing the effect of anthelmintic drugs at molecular level in
the helminth parasites. Carbohydrate plays a major role in energy
metabolism of helminths, which require a continuous supply of
energy (ATP), to motivate the complex metabolic processes
essential for their normal growth and development. Hence,
carbohydrate metabolism has been extensivly studied in helminths.
Since then many compounds have been screened for their
chemotherapeutic effects and the available literature has been
comprehensively reviewed by many workers ( Bando, 1951; Guilhon,
1968; Oakley, 1978; Van den Bossche,1978, 1980; Mansour, 1979;
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l l
Armour, 1983; Coles, 1983; Gutteridge, 1985; Bogan and Armour,
1986; Boray, 1986; Campbell, 1986 a; Prichard, 1978 , 1986;
Chappell, 1988; Subrahmanyam, 1987). Moreover many books such as
Chemotherapy of Parasitic Diseases ( Campbell and Rew, 1986),
Chemotherapy of Gastrointestinal Helminths ( Van den Bossche et
al., 1985 ) have been published and reviewed the available
literature.
Among the various compounds that have been tested so far,
the benzimidazoles, salicylanilides and organophosphorous
compounds are found to be quite effective against helminth
parasites. In the present work three anthelmintics were selected
namely mebendazole from the benzimidazole group, oxyclozanide and
rafoxanide representing the salicylanilide group.
BENZIMIDAZOLE COMPOUNDS: The benzimidazoles were introduced as a
group of safe and effective broad spectrum anthelmintics with the
discovery of thiabendazole by the Merck group in 1967. Further
Smith Kline and French introduced fenbendazole in 1967 and since
then many other international pharmaceutical companies have
brought a number of benzimidazole compounds like cambendazole,
mebendazole, oxibendazole, thiophanate, fenbendazole, albendazole
and oxfendazole on to the market.
MEBENDAZOLE: Mebendezole ( Methyl - 5 - benzoyl benzimidazole -2-
carbamate) was introduced as a new potent anthelmintic compound
by Janssen Pharmaceutica in 1971 as product No R 17635. In vitro
efficacy and therapeutic action of mebendezole against a number
of helminths was reported by varic is workers ( Chaia and Cunha,
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1 1
1971; Brugmana et al., 1971 and Banerjee et al.,1971). Van den
Boasche (1972) reported for the first time the biochemical effect
of mebendazole on a number of nematodes and cestodes both in in
vitro and in vivo conditions. A number of biochemical effects of
this drug have been demonstrated in different species of
helminths which together account for its broad spectrum
anthelmintic efficacy. De Nollin and Van den Bossche (1973) have
studied biochemical effects of mebendazole on Trichinella
spiralis larvae in vitro and reported that mebendazole inhibits
the uptake and /transport of glucose and increases glycogen
depletion in larvae. This drug also reduces the uptake of
fructose 3-O-methyl glucose, glycine, methionine, proline and
palmitic acid by Ascaris suum (Vanden Bossche and De Nollin,
1973). Recently Ahmad and Nizami (1991) has studied the effect of
mebendazole on the carbohydrate metabolism of Gigantocotyle
explanatum under in vitro conditions and concluded that
mebendazole inhibits the glucose uptake due to the inhibition of
phosphomonoesterases and the rapid depletion in endogenous
glycogen content may be due to stimulation of phosphorylase and
phosphoglucomutase.
Mebendazole induces many other effects on energy metabolism
in helminths like malate induced phosphorylation in Ascaris
mitochondria (Van den Bossche,1972) in viyo and in vitro decrease
of ATP synthesis and turnover of nucleotides (Rahman and Bryant,
1977; Bryant et al.,l976). A number of reports indicate that
mebendazole binds to mammmalian tubulin , the constituent protein
of microtubules (Kohler and Bachmann, 198 0, 1981; Ireland' et
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13
al.,1982). Kohler and Bachmann (1981) demonstrated that this drug
could inhibit ( H) colchicine binding to nematode tubulin in
competitive manner. Inhibition of microtubule system might affect
motility, absorption, secretion and enzyme activity in parasites.
Further,invitro inhibition of acid and alkaline phosphatses,
glucose-6-phosphatase and 5' nucleotidase was reported due to
mebendazole in Avitellina lahorea (Ahmad and* Nizami,1987).
Mebendazole has no effect on hexokinase activity of the helminths
while phosphorylase and phosphoglucomutase are affected by this
drug, which ultimately causes the depletion in glycogen content
and inhibits glucose uptake (Van den Bossche, 1972; Ahmad and
Nizami, 1987).
Biochemical changes caused by mebendazole in helminth
parasites have also been investigated histochemically in
order to support biochemical studies. Progressive time related
morphological changes in cestodes and nematodes after treatment
of the host with mebendazole have been demonstrated by electron
micrscopy ( Borgers et al.,1975; Verheyen et al., 1976).
Ultrastructural changes in the encysted Trichinellla larvae,
during and after treatment of the rat with mebendazole have been
reported (De Nollin et al., 1974). Degenerative changes were also
observed in the immediate infected host muscles like oedematous
swelling and intracellular vacuolization while loss of glycogen
was found in larvae cells. Borgers and Verheyen (1976) and
Verheyen et al. , (1976) have also observed tegujient. . changes in
Hymenolepis. nana and Taenia, taeniformis due to drug action and
suggested that disrurMon of microtubule formation may pre snt
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the outward movement of secretions from the subtegument to the
apical tegument.
Besides these effects in vitro trials of mebendazole have
also shown inhibitory action in the egg development, increase in
the rate of egg out put (Banerjee et al.,1971; Banerjee and
Prakash, 1972; Ahmad et al., 1987), inhibition of the intestinal
secretions (Atkinson et al. , 1980) and inhibition of acetyl-
cholinestrase secretion In helminths (Watts et al., 1982).
Further, mebendazole is also reported to reduce the rate of egg
production in benzimidazole resistant nematodes at normal
therapeutic doses (Kelly et al., 1975). Many field trials and in
vivo experiments have revealed that mebendazole has a broad
spectrum effects against helminth parasites in both domestic and
wild animals. Fernando and Denham (1976) have studied the
efficacy of mebendazole under i,n vivo conditions against encysted
muscle larvae of Trj.chinella spiralis in mice while Kelly et
al.(1975) have reported the effect of particle size on
anthelmintic efficacy of mebendazole against Nippostongylus
brasiliensis in the rat.
SALIC}YLANILIDES: Among the salicylanilides " Diaphene" and
"Hilomid" were possibly the first compounds used as flukicides
(Boray et al.,1965; Lienart, 1963;). Salicylanilides and
substituted phenols show variable activity against helminths, but
are usually most active against blood sucking worms ( Stampa and
ferbi nche, 1961; Horak, 1962; 1964; Boray, 1969; Sinclair and
Prichard, 1975). These compounds generally bind with the plasma
proteins and cause metabolic disorders in the parasites. Lee
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15
(1973) reported that fasciolicidal activity of salicylanilides in
sheep was dependent on the extent to which they persist in the
plasma. Salicylanilides and substituted phenols are also potent
uncouplers of oxidative phosphorylation (Williamson and Metcalf,
1969). Scheibel et al.(1968) demonstrated uncoupling of oxidative
phosphorylation in tape worm due to niclosamide under in vitro
conditions. Oxyclozanide, nitroxynil, rafoxanide and nitroscanate
uncouple oxidative phosphorylation in F. hepatica and A^
lumbricoides (Corbett and Goose, 1971; Van den Bossche, 1972 and
Cornish and Bryant, 1976). Further, Cornish et aO,. (1977) and
Prichard (1978b) suggested evidence of uncoupling during in vivo
trials.
OXYCLOZANIDE: Oxyclozanide is one of the most potent
salicylanilide compound active against trematodes and cestodes.
Broome and Jones (1966) have reported the therapeutic activity of
oxyclozanide against Fasciola sp. They found that this drug is
absorbed and binds to plasma proteins and transformed in liver to
the anthelmintically active glucuronide and concentrated in bile
in the environment of adult fluke.
A number of successful trials have been carried out on the
efficacy of this drug against F. hegatica, H. diminuta, H.
microstoma. Stilesia hepatica and few amphistomes (Broome and
Jones 1966; Wally, 1966; Harrow; 1969; Foreyt and Todd, 1973;
Hopkins et al., 1973; Corba et al., 1976; Georgiev and Gruev,
1979; Denev et al., 1985). Some in vitro studies have also been
accomplished on the biochemical and metabolic effects, resulting
into the inhibition of malate dehydrogenase o*. F, hepatica , F.
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qlqantlca, Fagciolopais bualci, and Paramphistomum explanatum. (
Lwin & Probert, 1975; Coles et al., 1980 and Probert et al.,
1981) and uncoupling oxidative phosphorylation (Corbett & Goose.
1971). Further, Edwards et al., (1981) also reported the effect
of oxyclozanide on the metabolism of F. hepatica.
Further reports are also available on the efficacy of
oxyclozanide alone or in combination with levaraisole against both
mature and immature paramphistomes (Schielhorn Van veen and
Bida,1975; Chhabra et al.,1977; Prasad, 1983). Oxyclozanide has
been formed highly effective against the pathogenic immature
forms in sheep or goats at a dose rate of 15 mg /Kg body weight.
Many reports from Eastern Europe and from USSR confirm the
efficacy of the drug against the adult parasites in both sheep
and cattle ( Denev et al, 1985). It appears that in an outbreak
of acute paramphistomiasis in calves oxyclozanide may be the most
suitable drug.
RAFOXANIDE: This salicylanilide is highly effective against adult
and to a lesser extent against immature F. hepatica (Lucas, 1967;
Boray and Happich, 1968; Ross, 1970; Armour and Corba, 1970;
Annen et al., 1973; Boray et al^ 1973; De Keyser, 1980). This
drug has been found active against some nematodes particularly i
Haemonchus contortus and nasal mj asis of sheep Oestrus ovis
(Lucas, 1967; D'souza et al,. , 1992) Increased dose rate from the
normally recommended was found highly active against fluke aged 4
weeks or older (Armour and Corba, 1970; L wards and Parvy, 1972).
Fasciolicidal activity of the rafoxanide is due to retention
ability in F asma (Lee, 1973). The half life is about 4 days and
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17
it binds to plasma proteins with a high affinity. Cornish et al.
(1977) and Prichard (1978b) provided the evidence of uncoupling
of oxidative phosphorylation in vivo. Some i,n vitro studies have
also been accomplished on the biochemical and metabolic effects,
resulting in the inhibition of succinate dehydrogenase and
fumarate reductase system in F. hepatica ( Duwel and Metzger,
1973; Metzger and Duwel, 1973; Coles and East,1974).
The foregoing review of the literature, indicates that all
the above mentioned compounds show anthelmintic activity in one
way or the other. Different parameters have been used by the
various investigators for the assessment of their efficacy. In
vitro studies such as biochemical effects, ultra structural
damages and electrophysiological changes on the surface of the
parasites also provide informations about the mode of action of
a particular drug which ultimately forms the basis of in vitro
trials.
In recent years the electron microscopic evaluation of
damage have also been used as a powerful tool to study the actual
site of morphological disorganization due to drug action.
Erasmus (1970) pointed out through scanning electron microscopy
the physiological importance of the tegumental surface of the
digenetic trematodes in the host parasite relationship. Recently,
Dunn et al. (1987) and Mattison et al. (1992) have reported some
specialized structures of tegument of the amphistomes under study
and suggested their possible role in establishment of parasitic
mode of life and possible chemoreceptive function. Drug induced
topographical changes have been reported by Probert et al. (1982
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1 ••]
in Moniezla. expansa treated with oxfendazole and Praziquantel in
vitro. Ahmad et al. (1987) have also reported some topographical
changes and observed that oxyclozanide produce most pronounced
damage in G. explanatum while mebendazole produced least effect.
Pax et al. (1978) have reported that praziquantel causes
contractions in the musculature of S. mansoni and showed that
this drug decreased the influx of Na and Ca . They suggested that
spastic paralysis In the worms may be related to depolarization
of the cells, elicited through inhibition of Na"- K* ATPase
function. Coles (1979) suggested that in Schistosoma.mansoni
praziquantel opens pores in membranes and permits a rapid influx
of Ca*"*" either directly or indirectly through an effect on the
influx of Nat
Mehlhorn et al. (1983) have also investigated the effect of
praziquaentel on some human trematodes namely Clpnorchis sinensis
.Metaqonimug yokoqawai, Qpisthgrchis viyerrlni. Paragonimus
westermani and S. japonicum and reported that all flukes exposed
to the drug contracted and showed tegumental alterations such as
papillae and smoothing of surface. In vivo effects of amasconate
have been studied by Voge and Bueding (1980) and reported
tegumental swelling in S. mansoni. Kohn et al. (1982) reported in
vivo effects of oxamniquine on the tegument of male S. mansoni
and found bubble like lesions.
Ultrastructural studies on helminths have revealed that the
tegument is metabolically active and plays an important role in
transmembranosis of sugars, amino acids and other substances (see
reviews by Lumsden, 1975 ,- Erasmus, 1977; Threadgold, 1984). The use
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13
of radiolabelled subifc'ates in physiological studies of helmintHs
has provided a conclusive evidence that tegument play a vital
role in uptake mechanism. Further, by the use of this technique,
it is also possible to work out actual mode of uptake, site of
incorporation in the organelles or in the complex biochemical
moities (Hanna, 1975, 1976, 1980; Specian and Lumsden,1981)
The autoradiographic examination of labelled hexoses and
amino acid incorporation shows that a significant amount of
radioactivity incorporated into macromolecules in the tegument
and various other organs of trematodes. Monosaccharide absorption
has been studied mainly in F. hepatica and Schistosoma spp. and
the evidence suggest that hexose uptake occurs through the
tegument rather than oral orifices as gut involvement was
precluded by ligation of the pharynx (Mansour, 1959; Isseroff and
Read, 1974) or by the exclusion of the mouth from the test medium
(Roger and Bueding, 1975). Similarly Parkening and Johnson (1969)
have also observed the absorption of tritiated glucose by the
tegument and not by the intestinal caeca in starved or unstarved
adult Haemonchus medioplexus and Gorgoderlna spp. Recently Khan
(1991) have studied glucose transport kinetics in gut ligated
amphistomes under study and reorted that uptake of ^H-glucose
being taken up through the tegument.
In addition to this, a number of known drugs affect the
motility of the worms. Such motility recording , under in vitro
conditions, seems to be an effect! • e, quick and economic method
for screening a large number of drugs. Prob^rt et al.(198l)
studied visually the motality of the worms in Vitro in presence
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. 1 )
of five flukicides including oxyclozanide in three trematodes of
veterinary importance namely F. gigantlca, Fasciolopglg buski and
Paramphistomum explanatum. Another drug diarafenetide also caused
paralysis in F. hepatica as observed visually by Rew et
al.(1983). Oxyclozanide is the only drug which has been tested
for amphistomes, while other drugs under study, have been
completely neglected. Recently, Ahmad and Nizami (1990) have
examined the in vitro effect of certain anthelmintics on the
motility of G. explanatum. It was observed that mebendazole
induces irreversible spastic paralysis, where as fenbendazole
causes irreversible disturbed activity. Metrifonate produced
rapid decrease in muscle tone leading to flaccid paralysis.
Oxyclozanide also induced immediate suppression of motility with
increasd muscle tone leading to irreversible spastic paralysis.
Further Ahmad and Nizami (1992) reported that oxyclozanide
induces time dependent irreversible spastic paralysis in four
amphistomes including G. crumenifer.
It is now an established fact that when an infected animal
is treated with anthelmintic drugs through any route of
administration, the drug molecules first come in contact with the
surface of the parasite. There after binding with an appropriate
receptor, the drug molecule are trasferred from the surface to
inside the cell and produce either morphological or biochemical
effects. This shows that parasite surface is a primary site for
drug action. With this assumption the present study was designed
to examine the effect of some known anthelmintics on the surface
plasma membranes of the amphistomes. Any adverse effect on the
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11 biochemical configuration of the plasma membranes would certainly
influence the host-parasite relationship, which is the most
delicate and harmonious relationship in the successful
establishment of the parasite. Any disruption in this
relationship could be exploited for the control of infection.
This seems to be an important and appropriate rational approach
for the parasite control. ^
However, the other aspects of chemotherapeutic control like
toxic or side effects can not be ignored. Many pharmacological
agents induce a number of side effects and among these
immunotoxic effects are of prime importance.
IMMUNOPHARMACX>LOGICAL STUDIES
The main role of the immune system is to sustain the host
defence mechanism and to maintain homeostasis of the host.Two
main categories of unwanted consequences are produced by the
interaction of pharmaceutical drugs and the immune system (i)
alterations of host defence mechanism with either impaired or
enhanced normal immune response (ii) induction of qualitatively
abnormal immune response. The former effect is likely to
represent a direct toxic effect on the immune system while the
latter is associated with clinically significant unwanted
reactio-ns. Unfortunately immunotoxicologists largely focus their
attention on the immunodepressive activities, however immune
enhancement or immunestimulation should also be given equal
emphasis.
There are few scattered studies which have been carried out
on the chemotherapy of parasite infections and its relationship
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22
with the immune status of the host. Since the disciplines of
chemotherapy and immunology have developed independently and
phrmacologists have mainly focusssed their attention on greater
parasiticidal properties in the drug development rather than
their interaction with the immune response, therefore an inter
disciplinary approach is required to achieve this goal. Targett
(1985) (Comprehensively reviewed the available literature on
chemotherapy and immune response to parasitic infections and
grouped the various reports in following four categories:
(1) Immune dependence of antiparasitic chemotherapy
(2) Immunodepression by chemotherapy
(3) Chemotherapy and induction of immunity
(4) Immunopotentiation and chemotherapy
The only comprehensive review available on the immunotoxic
effects of drugs is by Descotes (1988). There are various anti
parasitic agents which are currently used as therapeutic or
prophylactic agents in order to control parasitic diseases which
aa/ersely affect the immune system leading to susceptibilty for
subsequent infections. Unfortunately, most studies of this nature
have been carried out on protozoans while helminths remained some
what neglected. The toxicity of the drugs is measured by
examining both cellular and humoral immunity. The various
parameters which have been used to test the toxicity have been
reviewed by Berlin et al.(1987) and Luster et al. (1982) and can
be grouped into three major categories:
Hi Histopathology of immunocompetent organs includes lymphoid
organs, thymus and various lymph nodes. Histopathology in
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Z3
broad sense may include T and B lymphocytes, bone marrow cells
and their differentiation from a common multipotent stem cell.
The various aspects can be assessed by colony forming test or
Immunocytochemistry.
(2) Humoral immunity is another aspect to assess the immune
system which includes the level of immunoglobulins mainly IgG and
IgM using different immunoanalytical procedures like ELISA,
immunoblotting, lEP etc. Besides Igs level, haemagglutination,
plaque forming cell technique have also been used.
(3) Cell Mediated Immmunity. This can be assessed by classical
delayed type hyper sensitivity response to antigen, migration
inhibition test, lymphocyte proliferation assay using various
mitogen and lymphocyte toxicity.
In recent years parasitologists have accepted this
interdisciplinary approach of research. A number of papers have
now appeared in which the combined role of drugs and immune
system have been investigated. In the past decade substantial
evidence for the immune dependence of chemotherapy has
accumulated. Immunosuppression is known to reduce the efficacy of
chemotherapy in several parasitic diseases such as rodent malaria
(Lwin et al. , 1987), trypanosomiasis (Frommel, 1988),
onchocerciasis (Bianco et al.,1986) and schistosomiasis (Doenhoff
and Bain,1978). It is also well documented that severe cases of
strongyloidiasis in the immunocompromised individuals often fail
to respond to anthelmintic treatment, and repeated drug treatment
is necessary to obtain a complete cure(Scowden et al.,l978;
Weller et al.,1981; Shelhamer et al. , 1982-, Korgan et al. , 1986)
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Although the immune factor involved in influencing the
drug efficacy is not yet determined, it has been reported that
antibodies are synergistically involved in enhancing the efficacy
of drug against schistosomes and malarial parasites. When immune
sera was administered simultaneously with the drug, greater
degree of cure have been achieved in immunosuppresed hosts in
schistosomiasis and malaria inffctiorv (Brindley and Sher, 1987;
Targett, 1985). Cyclophosphamide, the nitrogen mustard derivative
when administered to animals before antigenic stimulus has been
shown to reduce selectively the number of B lymphocytes in
lymphoid tissues while having little effect on the T lymphocytes
(TurJc et al. 1972; Turk, 1973). The B cell response as measured by
antibody production to sheep red cells appears to be markedly
impaired by such pretreatment with cyclophosphamide (Smith and
Hughes, 1974), while T cell function is actually increased (Turk
et al., 1972; Kerkhaert et al., 1974; Lagrange et al. 1974).
Vickerman et al. (1976) observed in T. brucei infection that when
cyclophosphamide was given on 'hird day after infection, a high
non relapsing parasitaemia developed in treated animals. Such
immunological variations have a direct impact on epidemiological
studies as suppression of skin t6st in filarial patients treated
with DEC was observed (Sawada et al.,1968; Katiyar et al., 1974
and Murthy et al.,1978) . Keuffer (1976) observed changes in
haemagglutinating antibodies even after a single dose of DEC. In
Litmosoides carlnii infection when orms are transplanted
intraperitonially the immune status shows variation after DEC
administration (Misra et al.,l982). Effect of DEC on <3MI response
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^5
wag also investigated by Piessens et al.(1981), Mistry and
Subrahamanyam (1986). These findings indicate that immune status
of filarial subjects may be altered as a result of DEC
treatment.These studies also indicate that the authenticity of
sero-epidemiological results become doubtful if the host had been
treated with antifilarials previously. Contrary to these
observations Bekhti et al.(1977) reported increased level of
circulating immune complexes in patient's sera after mebendazole
chemotherapy, but specific IgE level decreased. The parasitocidal
activity of mebendazole on the larval stages of E. granulosus
have been observed in experimental animals (Eclcert et al.,1978
and Schantz et al. 1982) . Further, Muller et al,. (1982) and
Schantz et al. (1982) tested the efficacy of this drug in human
cystic and alveolar echinococcosis. In order to assess the effect
of this drug on antibodies level a study was carried out on
patients undergoing prolonged treatment with mebendazole and
observed a decreased level of antibodies as well as total serum
IgE concentrations (Woodtli et al.,1985). In E. granulosus
infection decreased antibody titer was also observed following
chemotherapy (Dessaint and Capron, 1982; Schantz et al.,1982;
Loscher, 1983).
In another study Gottstein et al^ (1984) investigated
parasite specific immunoglobulins by ELISA in a number of
patients of E. granulosus before and after mebendazole treatment.
In majority of cases IgG decreased and levels of IgA and IgE
gave inconsistent results. However, in progressive disease
Condition the reverse tendencies were observed in the level of
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• J "*
Igs. These authors are unable to explain the cause of decrease in
Igs under chemotherapeutic pressure. OzeretskovsJcaya et al. (1979)
suggested that such effects may be a consequence of reduced
antigen release or to be a toxic effect of the drug on immune
system.
Nolla Panades et al. (1980) have reported after treating otie
patient with multicystic liver cyst that antibody level declined
to negative values following chemotherapy with mebendazole while
Kovrova et ai. (1979) have suggested that mebendazole in
experimental echinococcosis is responsible for a reduction of
specific humoral immunity and a stimulation of specific cellular
immunity.
Kamath et al. (1985) also observed a decline in antibody
titer in A. ceylanicum infected hamsters following mebendazole
therapy, whereas levamisole treated hamsters could restore the
titre later because of its immunopotentiating action (Janssen,
1976). Similarly Khan et al,. (1991) have also investigated
alterations in immunological response before and after
chemotherapy in hamsters infected with A. ceylanicum. Humoral
response assessed by ELISA depicted gradual depletion in antibody
titre following treatment with mebendazole, albendazole and
pyrantel pamoate.
In addition to this, such combined effects of drugs on the
immune system and their efficacy have also been carried out on
various species of nematodes particularly on Nippostrongylus
brasiliensis in which DEC either inhibits sJcin test or
significantly increase susceptibility of subsequent infection. In
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L i
both the cases there must be obvious decline in the antibody
titer ( Katiyar et al. , 1974, 1978, 1985; Chatter jee et al.,1976,-
Murthy et al., 1978). In another filarial worm, D. immitis marked
increase in ELISA titers has been observed subsequent to an
initial thiacetarsamide treatment and the titre gradually
decreases in drug treated dogs (Grieve and Knight, 1985).
Similar changes in immune system have also been reported
from S. mansoni infection by using fluorescent antibody detection
techniques (Ambroise-Thomas et al., 1970). In Trichinella spp.
infection antibody titer show changes following anthelmintic
treatment (Corba et al., 1977). Further it has been observed
that administration of compound 48-80, made the immune mice
susceptible for S. mansoni infection (Ogilvie, 1974 and Dean et
al.,1976).
Such immunosuppressing effects of post treatment period are
generally being c( sidered as major hurdles in the interpretation
of sero epidemiological data of a particular endemic region. In
Fasciola hepatica. stage specific antigens (Tl and T2 bodies)
have also been tested for their antibody response following
diamphenithide treatment (Hanna et a1.,1982). They observed that
the antibody titer against Tl antigen was unaffected by the drug
treatment from 3 to 6 weeks, however, the T2 antibody response
was some what reduced. This shows that types of antigen also play
a significant role in antibody titer. Benex et al. (1973) also
observed a significant reduction in the antibody titer following
anthelmintic treatment in F. hepatica infection in sheep. Some
other workers have also reported that antibody titer drop
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z.'
dramatically in rabbits and rats infected with F. hepatica
following rafoxanide treatment (Hillyer and Llano de Diaz,1976
and Hillyer and Santiago de Weil, 1979). Similarly in another
trematode infection, reduction in antibody titer and complement
activity was noticed following rafoxanide or ivermectin treatment
in sheep infected with Hasstilesia (Daugalieva, 1986). Besides
these reports malarial infection and chemotherapy have also been
investigated on these lines. Quinine and primaquine can depress
the lymphocyte response to mitogens such as PHA and to various
antigens (Thong and Ferrante,1978; Gold et al., 1979). Mefloquine
depressed antibody formation but did not affect delayed type
respoBse (Thong et al,. , 1979). In addition to this chloroquine
induces suppression of lymphocytes just like quinine and
primaquine under in vitro condition (Hurvitz and Hirschorn,1965).
It has been reported that chloroquine stabilizes lysosomal
membranes interfering with processing of antigen (Hurvitz and
Hirschorn, 1965 and Lee et al.,1982). Further, the concentration
of chloroquine 0.8 ug/ml is also an important factor in inducing
some immunotoxic effects as interference with release of
interleujcin I from monocytes and thus inhibits generation of
antibody producing cells (Salmeron and Lipsky, 1983).
In some other protozoan infection like Giardia. Entamoeba
•and Trichomonas metronidazole suppresses the CMI reactions, such
as granuloma formation. The effect can be enhanced when it is
simultaneously administered with antigen (Grove et al.,l977).
Comparatively a new drug Cyclosporin A (CsA), a fungal derivative
was developed for clinical use because of its immunosuppresaive
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properties. It reversibly inhibits transformation of stimulated
T cells, suppressing both humoral and cell mediated immunity.
This drug is also reported to have antimalarial activity
(Thommen-Scott, 1981). Besides its immunosuppressive and
antimalarial activity, the anthelmintic properties and
immunomodulatory nature have been comprehensively reviewed by
Chappell and Wastling (1992). Because of its profound
immunomodulatory nature andantischistosomal action of CsA is
mediated via an augmented immune response although the usual
effecs of CsA on the immune system are down regulation of T cell
related events (Thomson et al., 1986). CsA can potentiate the
immune response to non parasite antigens under certain conditions
(Aldridge and Thomson, 1986; Webster and Thomson, 1987) and
augmented pulmonary granuloma formation around the schistosome
egg (Metzger and Peterson, 1988). The small number of published
reports on the action of CsA on cestodes reveal that the drug is
antiparasitic against some species while it may be
immunosuppressive for other infecti ns.
Having established that the effectiveness of many drugs can
be increased by the immune response it is logical to try
combining drug treatment with compounds that enhance immunity.
Among different anthelmintics only levamisole was reported to
have immunostimulating action (Merluzzi et al. ,1976; Renoux and
Renoux, 1977). It has been used for chemotherapy of helminths
either alone or in combination with other drugs.
It is evident from the foregoing review of the literature
that, the mode of action of various anthelmintics have been
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studied by a number of workers but no generalization can be made
on the basis of present status of our knowledge, because it is
often stated that a particular drug may have multiple modes of
actions. Also the same anthelmintic may behave differently in the
taxonomicaily different group of parasites, thus pointing the
necessity of studying each member separately to investigate the
mode^ of action of various anthelmintics and the possibility of
their broad spectrum efficacy.During the course of this study it
was proposed to investigate the effect of mebendazole,
oxyclozanide and rafoxanide on the membrane bound marker enzymes
of G. explanatum and G^ crumenifer inhabiting two different
habitats. Furthermore, effects of these drugs on the glucose
uptake was also monitored. Oxyclozanide is a known flukicide and
is frequently used, hence, it was decided to investigate its
effect on immune system of laboratory animals.
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31
MATERIALS AND METHODS
PARASITE OOLLECTTION: Active and adult Gastrothylax crumenifer and
Giqantocotyle explanatum were collected from fumen and liver
respectivly from Indian water buffalo Bubalua bubalis slaughtered
at local abbatoir. The worms were bVought to laboratory
expeditiously and washed in three changes of Hank's balanced salt
solution (HBSS) without glucose premaintained at 37+2°C.The worms
were immediatly used for isolation of surface plasma membrane and
for other studies.
ISOLATION OF SURFACE PLASMA MEMBRANE: The method used for
isolation of surface plasma membrane was essentially the same as
described by Rahman et al.(1981a) with some modification
suggested by Khan (1991).The membrane disrupting solution (MDS)
constitutes 1* saponin in HBSS pH 7.8.The MDS solution was always
prepared fresh, cetrifuged for 16,000xg for 60 min and the
supernatant was filtered and stored at S C until used.
Fourty worms of each species were rinsed quickly in HBSS,
premaintained at 37+2 "C and transferred to 100 ml flask
containing 20 ml MDS at 4*0. The flask were kept for continuous
shaking on electrically driven shaker maintained at 4 "c.The
surface plasma membranes were removed by continuous shaking (90
cycles per min) for 20 min. After shaking, the MDS containing
worms were tansferred in a 25 ml stoppered tubes and vortexed on
cycloraixer at high speed for 30 seconds.The carcasses of the
parasites were removed and the contents were pas&ea through
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'6
Worms + MDS C 1% Saponin ) .
Shaken at 4 C, 20 min. (90 Cycles/ min.)
Vortex (30Sec.)
Carcass Discarded
Pellet Discarded
MDS Surface Plasma Membrane
Centrifuged at 1500j5.g,15 min.
Supernatant i
Centrifuged at 16,000 X g. 30 min.
Pellet S u p e r n a t a n t "^resuspended in Tris HCl.pH 7.4 Discarded I
Centrifuged at 16,000 X g, Ih
Supernatant Discarded
Pellet
TEM Enzyme quantitation F i g . 1: Flow diagram for the isolation of surface plasma membrane .
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3 3
coarse sieve.
The filtrate containing maximum foam and plasma membrane
fragments were kept at 4*0 until used for centrifugation.
CENTRIf^GATION: The MDS containing surface plasma membrane were
first c ntrifuged at low speed (1500 x g) for 15 min in a
refrigerated centrifuge (I E C , 25) at 4 ''c.The pellet which
contained mainly debris was discarded and the supernatant was
further centrifuged at high speed at 16,000 x g for 30 min in
BecJcman L8-55 Ultra Centrifuge. The resultant pellet was
resuspended in 0.1 M Tris- HCl buffer (pH 7.0 at 4 "o and
centrifuged again at 16,000 x g for JO min, the buffer was
discarded. In order to wash the pellet and remove the traces of
detergent, the pellet was resuspended in Tris- HCl buffer and
centrifuged for 1 hr at 16,000 x g. After washing, the final
pellet was dissolved in a known volume of 0.1 M Tris - HCl buffer
(pH 7.4) and sonicated for 30 sec under chilled conditions (4 °C)
by using a 5 mm probe of ultrasonic disintegrator ( Ralsonic,
India).
TRANSMISSION ELECTRON MICROSCOPY: The membrane pellets, recovered
after the treatment of detergent for 20 min, were fixed for 3 h
at 4 °C in 4X (v/v) glutaraldehyde in 0.1 M cacodylate buffer
containing 3* sucrose (w/v) and 0.015* CaCljCw/v). After the
primary fixation the pellets were washed three times in buffer
containing sucrose and CaCl^ . Thereafter,the pellets were post
fixed in 1 % (w/v) osmium tetroxide for l h, dehydrated in graded
sereis of acetone to propylene oxide and embedded in spurr resin.
Ultrathin sections were cut on LKB -IV ultramicrotome.
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• I r
3';
collected on bare copper grids and double stained with uranyl
acetate and Reynolds lead citrate sections were examined using
JEM 100 CX electron microscope at various magnifications.
PROTEIN ESTIMATION: The total protein content in isolated surface
plasma membrane fraction was determined by dye binding method c
Bradford (1976) as modified by Spector (1978) using bovine serum ft-
albumin (BSA) as standard.
PREPARATION OF HOMOGENATE: Adult worms were collected , washed in
two changes of HBSS, blotted and weighed. Ice cold 0.25 M sucrose
was added to give 10* (w/v) extract and then homogenized using
Potter-Elvehjm tissue homogenizer with teflon pestle kept in ice
bath. These homogenates were centrifuged at 1500 xg for 20 min at
4''c. The supernatant was stored at 4°C until required for enzyme
assay.
ENZYME ASSAY:
ACID PHOSPHATASE (EC 3.1.3.2): Acid phosphatase was assayed by
the method of Bergmeyer et al.(1974) using p-nitrophenyl
phosphate as substrate. The total reaction volume of 1.1 ml
comprised of 0.05 M acetate buffer pH 5 containing 8 mH p-
nitrophenyl phosphate (pnp), 0.05 M Tris- HCl pH 7.0 and protein
sample.The reaction mixture was incubated for 30 minutes at 37*'c.
Thereafter reaction was stopped by the addition of 8.9 ml of 0.02
N NaOH. The liberated p- nitrophenol was measured at 420 nm.
ALKALINE PHOSPHATASE (EC 3.1.3.1): Enzyme activity was determined
by the method as described by Bergmeyer (1974) using same
substrate as in acid phosphatase. Total volume of reaction
mixture was l.2 ml comprising o. 1 ml, 0.05 M. glycine-NaOH buffer
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3;j
pH 9.5 containing 5 mM substrate (pnp).
Protein sample and total volume was adjusted by Tris - HCl
buffer pH 7.0. Reaction mixture was incubated for 30 minutes at
37 'C and thereafter the reaction was stopped by the addition of
8.8 ml of 0.02 N NaOH. The liberated p-nitrophenol- was measured
at 410 nm.
Enzyme activity of acid and alkaline phosphatases was
determined by previously prepared standard calibration curve of
known concentration of p-nitrophenol. Activity of acid and
alkaline phosphatases was expressed as AJ gm p-nitrophenol
liberated / mg protein /min.
Y-GLUTAMYL TRANSPEPTIDASE (EC 2.3.3.22): The enzyme activity has
been determined according to the method of Szasz (1974).
The assay mixture (total volume 2.1 ml) contained 4.2 mM L-Y
glutarayl-p-nitroanilide, 52.5 mM glycylglycine, 10.5 M MgCl, 50
mM ammediol buffer pH 9.3 and protein sample (volume adjusted to
0.1 ml with ammediol buffer). Simultaneously, blank devoid of
protein sample was also prepared. The change in absorption
indicating the liberation of p-nitroaniline recorded as 405 nm on
Spectronic -1001.
The specific enzyme activity was expressed as n mole p-
nitroaniline liberated / mg protein / min.
AMWOSINE TRIPHOSPHATASE (Mg dependent) (EC 3.6.1.3): Enzyme
activity was determined as estimating release of inorganic
phosphorus in reaction mixture by the method of Ryre (1975) as
modified by Rahman et al.,(1981 b); Verna and Frati (1983). The
reaction mixture (total volume 2.2 ml) comprising of 0.05 ml
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protein sample , l ml of 92 mM Tris-HCl buffer pH 8.5, 0.5 ml of
5 mM MgClj. Total volume of the mixture was adjusted by Tris-HCl
buffer pH 7.0. After 10 minutes of equilibration of reaction
mixture at ST'C in shaking water bath the reaction was started
by the addition of 4 mM ATP (Disodium salt, Sigma Chemical
company U.S.A.). After 15 min of incubation at ST^C, the reaction
was stopped by the addition of ice cooled 10» TCA.Then the tubes
were centrifuged at 5000 x g and supernatant was assayed for Pi.
PHOSPHORUS ESTIMATKW: Phosphorus was estimated by the method of
Rouser et al.(1970).The samples were prepared as follows: 0.1 ml
of cone. sulphuric acid was added to 0.2 ml of supernatant /
standard solution and heated in boiling water bath for 2 min
then cooled at room temperature and 0.05 ml of 70% perchloric
acid was added and tubes were again boiled for 2 min. After
cooling, 2 ml of DDW followed by 1 ml of 1* ammonium molybdate
and 1 ml of 1% of ascorbic acid (freshly prepared) were added.
The contents were mixed by vortexing and heating at room
temperature at 70°C for 10 min. Finally after cooling the tubes
at room temperature, absorbance was read at 820 nm. Quantity of
phosphorus was determined by previously prepared standard curve,
using KH^PO^in range of 1-10 /U gm.
ANTHSLMINTICS TESTED:
1. MEBENDAZOLE ( Methyl-5-Ben2oyl-Benzimidazole-2-carbamate) was
dissolved in dimethyl sulphoxide (DMSO) because it is a safe drug
solvent for benzimidazo i.e compounds for in vitro studies (Ahmad
and Nizami, 1983). Final concentration of DMSO in each incubation
aixture was 6* . Controls were also run simultaneously in each
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3 7
study containing equal volume of DMSO.
2. OXYCLOZANIDE (2,2'-Dihydroxy-3,3'5.5'6-Pentachlorobenzanilide;
supplied by Imperial Chemical Industries Limited, Great Britain)
is an ethanol soluble compound, each concentration of this drug
was dissolved in ethanol. Controls were also run simultaneously
in each experiment. The final concentration of the ethanol was
approximately 3 \ in each incubation mixture.
3. RAFOXANIDE ( Supplied by Merck, Sharpe and Dohme Research Lab,
U.S.A ) was also dissolved in ethanol. The final concentration of
ethanol was approximately 3 * in each incubation mixture.
Controls were also run simultaneously with equal volume of
ethanol in each experiment.
To study the effect of drugs on membrane bound marker
enzymes of isolated surface plasma membranes of G. crumenlfer and
Q- explanatum each compound was added to the incubation mixture
directly to maintain the drug concentration in terms of nano
moles viz:
Mebendazole 202 nmoles in 150 /ul of DMSO
Oxycolzanide 112 nmoles in 70 AJI of ethanol
Rfoxanide 122 nmoles in 150 AJI of ethanol
Enzyme activity was then measured keeping the total assay
volume constant in case of each enzymes mentioned earlier. The
enzyme activities of the total parasite homogenate were estimated
in order to compare with enzyme levels of the membranes. The
statistical significance of the data was analysed by Student's t-
test (Sokal and Rohlf, 1981).
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3,]
^H- GLUCOSE UPTAKE BY LIQUID SCINTILLATION COUNTING:
Tnis study was carried out in rumen amphistomes, G.
crumenifer. Freshly collected worms were thoroughly washed in
HBSS and preincubated for 3 h without glucose. In order to study
the effect of different drugs ( mebendazole, oxyclozanide and
rafoxanide) on glucose uptake, three different concentrations of
these drugs were used in HBSS and the final radioactive glucose
concentration of l Ai Ci/ml (sp. activity 24 Ci/m mole) was
maintained in the incubation medium.
Four worms of same size were incubated in medium for 10 min
at 37-t-2*'c. Thereafter the incubated worms were immediately rinsed
and vortexed three times in ice cold saline without any labelled
material in order to remove adhered isotope. Thereafter the worms
were soaked on absorbent paper and kept for 24 hours in absolute
alcohol for extraction of incorporated material. To each
scintillation vial 8 ml scintillation cocktail containing 4 g PPG
(2,5-diphenyloxazole) and 50 mg POPOP (1,4-bis(phenyl-2-oxazolyl)
benzene,2,2-phenylene bis (5 phenyloxazole) per liter of toluene
was added and counted for 1 minute in Wallac, 1410 Liquid
Scintillation Counter, Sweden.
REVERSED ROCKET IMMUNOELECTROPHORESIS (RRIE): In order to monitor
the effect of oxyclozanide on the immune response the antibodies
were raised against the parasite. For this study the soluble
fraction of the total homogenate of the parasite was initially
usea to develop and to collect the hyperimmune sera of the
rabbit. Simultaneously the same rabbit was also given known
amount of drug. Tht concentration and other protocol details are
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t.)
given below. The quantification of the level of precipitating
antibodies against plasma membranes was carried out by using
reversed rocket Immunoelectrophoresis. This tecnique is
essentially the same as described by Laurell (1972) as modified
by Hoyeraal et al.(1975). In this method the antigen was mixed
with molten agarose gel and antisera were placed in the wells at
one edge of the gel. After electrophoresis the precipitation
bands in the form of rockets were analysed.
CiOLLBCrriON OF HYPER IMMUNE SERA: In order to develop hyper
immune sera against the soluble fraction of G. crumenifer,
freshly collected parasites were homogenized in 0.1 M phosphate
buffered saline, pH-7.4, centrifuged at 5000 x g and the
resultant supernatant was sonicated for 30 sec at 4 "C. Protein
was estimated by the method of Spector (1978) as described
earlier. The homogenate was emulsified with equal volume of
freund's complete adjuvant (Difco) and injected subcutaneously at
four different sites along the aide of spinal cord. Firrt
inoculation consists of 4 mg protein. Second and third boosters
were given at an interval of 25 days having protein concentration
2.5 mg/ml. Finally, hyper immune sera were collected after 10
days of third booster. For preimmunised sera, blood was collected
from ear marginal veins of the rabbit before inoculation of
parasite the antigen.
To determine the effect of drug on the humoral response,
oxyclosanide was orally administered (7mg/kg body wt) in two
experimental rabbits. First dose was given four days before any
antigen inoculation, following five consecutive doses at a
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"i I
regular interval of 10 days. Control sera used was without any
drug treatment.
PREPARATION OF ANTIGEUS: For the preparation of antigen surface
plasma membranes were isolated as mentioned earlier. The surface
plasma membrane pellet was used t;o test the antibodies raised
against the total homogenate of the same parasite. In order to
prepare antigen partially purified surface plasma membranes of G.
crumenifer were disrupted and sonicated in known volume of tris-
HCl buffer, pH-7,4.
PREPARATION OF GEL: For preparing an agarose gel, 0.9* agarose
(BDH-Electran) was molten in 0.05 M citric acid phosphate buffer,
pH-5.2 containing 2 mM calcium lactate. The antigen was diluted
by adding citric acid phosphate buffer and mixed with molten
agarose.The concentration of the antigen in gel was kept at 0.33
mg/100 ml gel. Thoroughly mixed antigen in molten agarose was
poured on a glass plate mfisuring 2x8x15 cm and having 3 mm
thickness of the gel. Thereafter the plate was kept at 4 "c for
30 min. A row of wells measuring 5 mm diameter was punched having
1 cm distance between each well on one edge of the gel plate. In
first five wells control serum (serum without drug treatment) was
applied with a concentration of 10 Ail, 20 /ul, 30 Ail, 40 AI1 and 50
Ail respectively without any further dilution. The next five wells
were loaded with drug treated undil :ed serum with the same
concentration as mentioned above. After loading the control and
test sera, electrophoresis was carried out at a ostant supply of
4 m amp current per well for 6 h without any cooling device. In
all experiments, gel without antigen was used as negative
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control. In order to form a bridge between cathode and anode
filter papers were aoalced in running buffer (0.05 M Citric acid
-Phosphate buffer) and placed on the immunoelectrophoreis
assembly so that the antisera containing well remain towards
anode.
STAINING OF GEL: After electrophoresis the gels were removed and
immersed in 5% sodium citrate solution for 10 min so that, false
precipitation may dissolve. Slides containing the gel were then
washed three times by immersing in physiological saline at room
temperature for at least 12 h. followed by immersion in distilled
water for 10 min to remove the adhering salts on the gel if any.
The slides were then wrapped inpreviously moistened filter papers
(Whatman No. 1) with distilled water and kept in thermostat
premaintained at 37 °C for drying. The dried gels thereafter
immersed in amidoblack staining solution ( consisting of 0.1 mg
amidoblack in 20 ml glacial acetic acid and made upto 1 litre
with distilled water). The slides remained dipped for 20 to 30
min until well stained arcs in the form of rocket appeaered. For
further refinement of the results, slides were rinsed with
distilled water and dipped in a clearing solution ( consisting of
ethyl alcohol - 400 ml, glacial acetic acid - 100 ml and double
distilled water - 1000 ml) for 30 - 40 min until a clear
background appears. The plates were photographed with a Canon AE-
1 camera using ORWO, 125 ASA, black and white film.
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RESULTS
ISOLATION AND PURITY OF THE MEMBRANES
The membrane pellets recovered after 2 0 min of treatment of
worms with saponin were subjected for transmission electron
microscopy in order to check the purity and degree of
contamination. It is evident from the electron micrographs of the
isolated membranes that membranes of both the amphistomes possess
basic structural organization which is similar to that reported
from other typical plasma membranes. They are basically
trilaminate, showing a bimolecular electron dense lipid layer in
between with associated translucent protein layer (Plate 3
and 4). At some places the Isoated membranes become puffed or
expanded, which may be due to hyposmatic effect of MDS indicating
that these membranes follow chemiosmotic hypothesis which has
been reported for other cellular membranes. No cytoplasmic
junctions or tight junctions were observed. At places dense
coagulated bodies have been noticed which give an appearance like
desmosomes but such bodies may also be due to the effect of
detergent. At higher magnification holes or pits like appearance
were noticed which could be due to the differential effects of
detergent on different lipid moities.
Thus it can be concluded from the present results that
saponin is a suitable detergent for the isolation of surface
plasma membrane of amphistomes and 20 rain treatment is optimum in
order to obtain appreciable quantity of membranes-with purity and
least contamination. In addition to this the vortexing time and
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Plate 3: Transmission electron micrographs of surface plasma membranes isolated after 20 min of saponin treatment: GastrothYlax crumeniier : a: showing membrane yield at low magnification (scale bar: 0.5 um). b: showing membrane structure at high magnification (scale bar : 0.2 um). Gigantqcotyle expJLanaturn -. c: showing membrane yield at low magnification, d: showing membrane stucture at high magnification, small arrow: trilaminate membranes, arrow heads: desmosome like appearance and circles: puffed membranes.
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44
Q ^>f - > . , W
'^y'Jir
"^••rlvf'
(5)2. J •«• »««^ {'
.-•ii^J / 3 ••> ^ ^ .
r-v:
4
A ^ • \
Wft
f
^
• -r
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43
Transmission electron micrograph of surface plasma membranes of Gastrothylax crumemfer after saponin treatment showing trilaminate structure ( arrow ), puffed membranes (circle),and pores (arraw Heads ).
PLATE- 4
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the centrifugation protocol adopted in this study was found to be
appropriate for optimum yield. However the transmission electron
microscopy alone does not provide any conclusive evidence of
membrane yield.
Therefore, various memi me bound enzymes have also been
used as parameter for the characterization of membranes. On
comparing the enrichment of membrane bound marker enzymes with
the enzyme activity of the soluble fraction of the worm
homogenate, it is evident from the present study that the level
of enzymes of membranes showed many fold increase than the total
homogenate. The degree of enrichment varies from 3 to 12 old
(Table. 1 and 2). The order of maximum enrichment of various
enzymes also show differences i,e acid phosphatase > alJcaline
phosphatase > Y-GTP > ATPase in G. explanatum. While in G.
crumenifer the sequence is different i.e. acid phosphatase >
ATPase > Y-GTP > alkaline phosphatase. It can be concluded from
these results that many fold enrichment of the known membrane
bound marker enzymes indicate that the specified isolation and
purification procedures are optimal for the yield of membrane
fraction.
QUANTIFICATION OF MEMBRANE AND SOLUBLE ENZYMES:
The quantitation of the primary marker enzymes of the
surface plasma membranes isolated after the treatment of the non
ionic detergent (Saponin) are summarized in Table 1 and 2. The
levels of acid phosphatase is higher than the alkaline
phosphatase in both soluble and membrane fractions of the
amphistomes. However, quantitative differences were observed in
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^ 7
Table 1. Enrichment of surface plasma membrane enzyme activities in ^ comparison with fresh worm homogenate ofG. explanatum
Enzymes Homogenate Membrane Fold Enrichment
ACPase
ALK Pase
ATPase°
Y-GTP*
3.700+0,
0.164+0,
0.466+0,
1.660+0,
.23
.02
.006
.39
45.45+3.
1.378+0.
1.540+0.
5.960+0,
. 15
, 15
.05
.77
12,
8.
3.
3.
.28
.40
.30
.59
* Values in ugm pnp liberate /mg protein/ min + SD o Values in mg Pi released/mg protein/min + SD • Values in nmole p-nitroaniline released/mg protein/min + SD
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43
Table 2. Enrichment of surface plasma membrane enzyme activities in comparison with fresh worm homogenate of G. crumenifer.
Enzymes Homogenate Membrane Fold Enrichment
ACPase 1.69+0.04 20.46+1.42 112.10
ALKPase 0.37+0.02 0.67+0.038 1.81
ATPase 0.24+0.01 0.95+0.07 3.95
Y-GTP 1.20+0.23 4.26+1.43 3.55
* Values in ugm pnp liberated/mg protein/min jt SD o Values in mg Pi released/mg protein/min + SD • Values in nmole p-nitroaniline released/mg protein/min + SD
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43
the level of the various enzymes in the two fractions of G.
explanatum and G. crumenifer.
In the soluble fraction among the various enzymes assayed,
acid phosphatase, Y-GTP and ATPase levels were found higher in G.
explanatum than G. crumenifer. While alkaline phosphatase
activity was greater in rumen amphistome than the liver
amphistome.
Whereas in the membrane fraction acid and alkaline
phosphatases, Y-GTP and ATPase levels were found higher in G.
explanatum than G. crumenifer. On comparing the enzyme data of
membrane and soluble fractions of both the parasites under study,
it is evident that with the exception of soluble alkaline
phosphatase all other enzyme levels were found higher in G.
explanatum.
EFFECT OF ANTHELMINTICS ON ENZYMES:
Some known anthelmintics belonging to benzimidazole and
salicylanilide group whose biochemical mode of actions are
known, were tested on the membranes of the araphistomes because
surface plasma membranes are being considered as primary target
of drug action.
MEBENDAZOEiE: Mebendazole is a known anthelmintic whose mode of
action is attributed with the blocking of transtegumental uptake
of glucose and binding with the tubulin of microtubular system.
It has been observed that in soluble fractions mebendazole has no
effect on the enzymes understudy in G. crumenifer (Table- 3)
while in G. explanatum with the exception of ATPase all other
•enzymes are significantly stimulated (Table- 5).
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Table 3. Effect of athelmintics on membrane bound marker enzymes of G. crumenifer ( soluble fraction)
bO
Drugs Cone, (nmoles)
ACPase ALKPase ATPase Y-GTP
NORMAL
MBZ
OXCL
RFZ
0.0 1.69+0.04 0.37+0.07 0.24+0.01 1.20+0.23
202 1.71+0.15 0.39+0.07 0.23+0.01 2.91+1.49 (p>0.05) (p>0.05) (p>0.05) (p>0.05)
112 1.96+0.04 0.40+0.015 0.23+0.002 0.95+0.36 (p<0.001) (p>0.05) (p>0.05) (p<0.001)
122 2.14+0.09 0.40+0.015 0.26+0.028 3.99+0.75 • (p<0.001) (p>0.05) (p>0.05) (p<0.01)
» Values in ugm pnp liberated /mg protein/min + SD o Values in mg Pi released/mg protein/min + SD • Values in nmoles p-nitroaniline liberated/mg protein/min + SD p value upto <0.05 is considered as significant.
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Table , 4. Effect of anthelmintics on membrane bound marker enzymes of G. crumcnifer ( membarane fraction)
Oi
Drugs
NORMAL
MBZ
OXCL
RFZ
Cone, (nmoles)
0.0
202
112
122
ACPase
20.46+1.42
22.13+1.09 (p>0.05)
21.64+1.84 (p>0.05)
22.28+1.48 (p<0.05)
ALK Pase ATPase Y-GTP
0.67+0.04 0.95+0.070 4.26+1.43
1.40+0.09 0.58+0.030 3.04+1.95 (p<0.001) (p<0.001) (p>0.05)
0.90+0.19 0.93+0.025 3.64+2.52 (p<0.05) (p>0.05) (p>0.05)
0.76+0.057 0.96+0.045 6.28+0.96 (p<0.05) (p>0.05) (p>0.05)
» Values in ugm pnp liberated/mg protein/min + SD o Values in mg Pi released/mg protein/min + SD • Values in nmoles p-nitroaniline/mg protein/min + SD p value upto <0.05 is considered as significant.
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Table 5. Effect of anthelmintics on membrane bound marker enzymes of G. explanatum ( soluble fraction).
Drugs Cone, (nmoles)
ACPase ALK Pase ATPase Y-GTP
NORMAL
MBZ
oxca.
0.0
202
112
3.70+0.231 0.164+0.020 0.466+0.006
4.120+0.304 0.270+0.016 (p<0.05) (p<0.001)
4.370+0.179 0.160+0.012 (P<0.01) (P>0.05)
RTC 122 4.450+0.302 0.174+0.014 (p<0.02) (p<0.001)
0.475+0.013 {p>0.05)
0.462+0.033 (P>0.05)
0.474+0.016 (p>0.05)
1.660+0.390
3.720+1.700 (p<0.05)
2.240+0.253 (P<0.05)
43.970+6.17 (p<0.001)
* Values in ugm pnp liberated/mg protein/min + SD o Values in mg Pi released/mg protein/min + SD • Values in nmole p-nitroaniline released/mg protein/min + SD p value upto <0.05 is considered as significant.
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Table 6. Effect of anthelmintics on membrane bound marker enzymes of G. explanaturn ( membrane fraction).
Drugs
NORMAL
MBZ
OXCL
RFZ
Cone, (nmoles)
0.0
202
112
122
ACPase
45.45+3.15
46.24+4.87 (p>0.05)
47.22+6.32 (p>0.05)
49.56+6,19 (p>0.05)
*
ALK Pase
1.38+0.15
3.58+0.93 (p<0.01)
1.33+0.13 (p>0.05)
1.43+0.14 (p>0.05)
0 ATPase
1.54+0.05
1.44+0.03 (p<0.05)
1.59+0.06 (p>0.05)
1.66+0.15 (p>0.05)
• Y-GTP
5.96+0.77
9.96+3.08 (p<0.05)
9.99+3.66 (p<0.05)
18.88+7.98 (p<0.01)
« Values in ugm pnp liberated/mg protein/min + SD o Values in mg Pi released/mg protein/min + SD • Values in nmole p-nitroaniline released/mg protein/min + SD p value upto <0.05 is considered as significant.
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b-t
In the membrane traction of rumen parasites mebendazole has
no effect on acid phosphatase and Y- GTP. Contrary to this,
highly significant stimulation was noticed in alkaline
phosphatase (Table- 4) where as ATPase of membrane origin
was inhibited in both rumen and liver parasites. I' G. explanatum
membrane bound alkaline phosphatase and Y- GTP are stimulated a
while on acid phosphatase no effect was observed (Table- 6).
These results reveal that some quantitative as well as
qualitative differences are noticed in the two fractions of both
the amphistomes inhabiting two different microenvironment.
OXYCniiOZANIDE: Oxyclozanide belongs to salicylanilide group which
is the most potent anthelmintic used for the control of immature
paramphistomiasis. The mode of action is to block MDH and
uncoupling of oxidative phosphorylation. In the present study,
oxyclozanide stimulates the acid phosphatase of the soluble
fraction, while had no effect on the membrane bound acid
phosphatase of both the parasites (Table 3, 4, 5 and 6).
Alkaline phosphatase of membrane origin of G. crumenifer is
stimulated while in both the soluble fraction as well as in the
membranes of G. explanatum this-drug is unable to induce any
effect (Tables 5 & 6).
The effect of this drug on Y- GTP is quite interesting in
liver parasite. The enzyme of both the fractions is stimulated
(Tables 5 & 6) while in the rumen amphistome Y- GTP of membrane
origin did not show any effect whereas Y- GTP of soluble fraction
was significantly inhibited (Tables 3 & 4).
Oxyclozanide had no effect on ATPase of both the parasite.
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J.)
in both the fractions. It is interesting that the differences
observed in the two fractions of both the parasites show some
degree of similarity and dissimilarity reflecting the variation
in the physicochemical properties of enzyme molecules.
RAPOXANIDE: Rafoxanide also belongs to salicylanilide group
which also interferes with energy generation mechanism of the
parasite and has greater affinity of binding with the plasma
protein in both the parasites with the exception of membrane
bound acid phosphatase of G. explanatum. This drug had no effect
on alkaline phosphatase of soluble fraction while producing
stimulatory effect on membrane bound alkaline phosphatase of G.
crumenifer and vice versa in G. explanatum (Tables 3 & 4, 5 & 6).
The Y- GTP is stimulated in both the fractions of parasites
understudy with the exception of membrane bound Y - GTP of G.
crumenifer. Rafoxanide did not produce any effect on ATPase like
oxyclozanide. This indicates that since these drugs belong to the
same group, have some degree of identity in their mode of action.
Besides this, some differences were also observed which can be
explained on the basis of physicochemical nature of the habitat
leading to differences in the membrane organization of the two
parasites.
EFFECT OF DRUGS ON^H - GLUCOSE UPTAKE: In order• to know the
possible role of the membrane bound enzymes in the
transtegumental uptake, the^H-glucose was used in the incubation
medi > in presence and absense of drugs. Different concentrations
of the drugs were used and the uptake was monitored by
scintillation count-lng.
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bG
3 Table 7. Effect of some anthelmintics on in vitro uptalce of H
glucose in G. crumenifer.
Drug
CONTROL
MBZ
OXCl
RFZ
Cone, (nmoles)
0.0
202 101 47.2
110 56 24
122 61 28.5
Glucose uptake (u moles/gm dry wt./lO min)
281.87
120.41 184.68 193.73
267.63 366.30 323.02
349.83 298.64 355.86
Values are the mean of 3 replicates
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Fig. 3: Effect of mebendazole on in vitro uptake of^H glucose by G. crumenifer. C: control
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5/
.a
•a
a \
O a
I
3
OR o u
300 -
260
200 -
160 -
100 -
6 0 -
C 47.2 101 202 Mebendazole concentration (nmoles).
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3 Fig. 4. Effect of oxyclozanide on in yit.ro uptake of H glucose
by G. crumenifer. C: control
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b8
400
.3 a ^ 300
a
o
a
IS V o
I—I O
200
100
C 110 56 24 Oxyclozanide concentration (nmoles)
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Fig. 5, Effect of rafoxanide on in vitro uptake of^H glucose by 5• crumenifer. C: control
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b.)
400
0 a o
rs
a
« o o 0 o
300
I 200 a
I M 4^
100
C 29 61 122 Rafoxanide concentration (nmoles).
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bO
The results of glucose uptake by G. crumenifer in presence
of different drugs are summarized in (Table. 7). After 3 h of
incubation in HBSS medium, G. crumenifer utilized different
amount of^H-glucose.
Mebendazole produced inhibition in glucose uptake. However,
the degree of inhibition depends on the drug concentration. It is
evident that the inhibition of glucose uptake is directly
proportional to drug concentration (Fig. 3).
It can be seen from the Fig. 4 that the lowest
concentration of oxyclozanide produced significant stimulation of
glucose uptake, and this stimulatory effect increases upto
second higher concentration. But highest concentration showed
significant inhibition of glucose uptake by G. crumenifer as
compared to control.
Rafoxanide also has a stimulatory effect on glucose uptake
t>y G. crumenifer but here a regular pattern was not observed. At
lowest concentration the glucose uptake was found to be maximum
and at next higher concentration it decreases. At highest
concentration glucose uptake again increases significantly. It is
evident from these results that all three concentrations of this
drug produced significant stimulatory effect by G. crumenifer in
vitro (Fig. 5).
It can be concluded from these results that benzimidazole
and salicylanilides show differences in their moete of action as
also observed in the inhibition and stiumulation of membrane
bound enzymes.
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b l
CONTROL ^ « • «
EXPERIMETAL M
Reverse Rocket Immunoelectrophoresis of Control and Experimental Sera Against Membrane Antigens of Gastrothylax crumenifer
PLATE 5
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Fig. 5. Diagrammatic representation of precipitation peaJcs following reversed rocket immuno-eletrophoresis.
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b ')
eOOOSee©^^ xiH 10 20 30 40 50 ' 10 20 30 AO 50
CONTROL EXPERIMENTAL
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.)
Table 8. Effect of Oxyclozanide on the antibody titer in rabbit.
Volume
10 ml
20 ml
30 mi
40 ml
50 ml
Height
Control
7.0 mm
10.0 mm
13.5 mm
14.5 mm
15.5 mm
of Peaks (mm)
Experimental
4.5
5.5
6.5
7.5
9.0
Specific Activity (Height/vol. of antiserum)
Control
0.70
0.50
0.45
0.36
0.31
Experimental Ii
0.45
0.28
0.22
0.195
0.18
-— *
ihibition
43
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EFFEX3T OF OXYCLOZANIM: QH CIRCULATING ANTIBODIES LEVEL BY REVERSE
ROCKET IMMUNOTLECTROPHORESIS: Finally an attempt has been made
to monitor the antigenicity of membrane protein and to workout
any possible immunomodulating role of oxyclozanide. The
immunomodulatory effect was monitored by asessing circulating
antiboty level through reversed rocket Immunoelectrophoresis.
The results obtained after the reversed rocket
Immunoelectrophoresis are shown in plate 5 and fig. 6. Two
distinct precipitation peaks were noticed when both drug treated
and untreated (control) sera were reacted with membrane antigens.
The ou r precipitation peak was sharp and prominent while the
second was found to be diffuse.This shows that two antigenic
components are present in the membrane antigen detected with
antisera raised against whole worm homogenate in both drug
treated and control animals.
It was observed that height of the rocket peaks were
directly proportional to the antibody concentration present in
the antisera loaded in antigen containing gel (Table. 8). On
comparing the immunoprecipitation pattern a gradual decrease in
the height of peaks corresponding to the antibody concentration
was depicted between drug treated and untreated animals. The
overall suppression in antibodies level were found to be 43% in
drug treated animal sera.
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DISCUSSION
It is evident that Saponin was found to be appropriate for
the isolation of surface plasma membranes of amphistomes and
appreciable yield of membranes was observed in 20 minutes of
detergent treatment with least contaminations.
Saponin has been frequently used for the isolation of
surface plasma membranes of Schistosoma sp, (Kusel, 1972;
Cordeiro and Gazinelli, 1979; Podesta & McDiarmid, 1982;
McDiarmid et al, 1983; NOel and Scares De Moura, 1986) and in
some cestodes (C •n et al, 1977; Oaks et al, 1977; Knowles and
Oajcs, 1979; Rahman et al. , 1981 a; Pappas, 1983; McManus and
Barrett, 1985 ). But the concentration and other conditions were
quite variable in the above mentioned studies. However, Saponin
has been reported to be the detergent of choice because at
concentrations below the critical micellar concentration, it has
the ability to enhance the ATPase activity (Stekhoven and
Bouting,1981). Therefore an optimum concentration of saponin
ci.e. 1 *) has been worked out by previous workers which has been
used in various helminths as well as in the present study.
Cain et al. (1977) and Oaks et al. (1977) have used 1%
saponin for 10 min for the iso-lation of membranes with intact
membrane bound enzyme activity. Rahman et al. (1981 a, b) have
also used 1% saponin in hypotonic and hypertonic MDS and obtained
comparatively more pure fractions of the brush border membranes
of H. diminuta with slight mitochondrial contamination. Similarly'
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bb
McManus and Barrett (1985) have obtained appreciable yield of
membranes in 1* saponin with 20 min detergent treatment of E
3E3Qyi03yS protoscoleces. All these workers have used 1 x 30 sec
shearing step on a bench top vortex mixer and differential
centrifugation procedures of varying time period. Purity of
surface plasma membranes isolated with saponin may be due to the
fact that saponin has been reported to produce differential
effects on the surface plasma membranes and on the basement
membrane of the tegument. The possible reason of low
contamination in membranes isolated with saponin treatment has
been suggested by Oaks et al.(l977) and Rahman et al. (1981,a).
They have reported that saponin does not appear to affect the
morphological integrity of the basement membranes of helminth
tegument, because a protein containing structure potentially
capable to interact with saponin (Assa et a1., 1973) and then
prevent the release of cellular components in the MDS.
The low contamination in saponin isolated plasma membranes
of amphismtomes may also be due to the ability of saponin to
interact with the hydroxyl roup of cholesterol as also suggested
by Kavanau (1965). Since cholesterol characteristically lowers
the fluidity of hydrocarbon chain in the hydrophobic region and
thus stabilizes membrane structure (Papahadjopoulas etal., 1974).
Therefore it may be possible that amphistomes when treated with
this detergent for short duration, saponin interacts with the
membrane cholesterol thus causing a reordering of endogenous
cholesterol resulting in structural weakening of the surface
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b?
plasma memrbranes and relatively mild vortexing can remove the
surface membranes.
Further Kavanau (1965) suggested that interactions between
the hydroxyl group of cholesterol and the unsaturated lactone
rin>:; of the saponin forms complexes, while Lucy and Glauert
(1964) believes that saponin creates pores in membrane and these
pores on fusion forms gaps. Such pores have also been noticed at
some places in isolated membranes of amphistomes in 20 min
saponin treatment by TEM. Appreciable quantity of cholesterol has
also been reported in membranes of amphistomes under study (Khan,
1991) .
The basic structure organization of the surface plasma
membranes of amphistomes understudy is essentially the same as
in the other helminths with the exception of schistosomes (see
review by Lumsden, 1975). The trilaminar structure has been
described by Singer and Nicolson (1972) for other cell memrbanes.
In addition to this the swelling at certain places in the
isolated membranes may be due to the osmotic stress of MDS. These
localized swellings on the isolated membranes suggest that these
portions may actually enclose various types of papillae reported
from the surface of these amphistomes (Dunn et al.,1987) which
may be involved in various physiological action like chemo/or
osmo reception.
Thus it can be concluded from the foregoing discussion that
saponin is the most suitable detergent for the isolation of the
surface plasma membranes of amphistomes with appreciable yield
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bS
and least contamination. Further the various membrane bound
enzymes could be used as markers for monitoring the purity of
these membranes.
The qualitative enzymatic differences were noticed in the
membranes of live and rumen amphistomes, which may be a
consequence of the parasitic adaption in physicochemically
different habitats. As McDiarraid et al., (1983) pointed out
that " in th studies of membrane purification it is necessary to
have a marker for identifying the purity of the membrane
fractions. This is usually accomplished by using enzymes unique
to the membrane being isolated." The presence of ^ cious enzymes
in the surface plasma membranes of amphistomes also suggests that
they follow the same enzymatic organization as in S. mansoni
inner bilayer as well as plasma membranes of other cell types.
Our results are in agreement with other reports available on
helminth parasites.
In the present study, acid and alkaline phosphatases, ATPase
and Y- GTP were found significantly high after the treatment with
saponin. It has been suggested that alkaline phosphatase could be
used as a marker for the tegument membranes. In helminths the
presence of alkaline phosphatase are often indicative of membrane
transport mechanism. Roberts et al. (1983) have used the alkaline
phosphatase as a marker for the surface plasma membrane of
Schigtosom. sp Further, Wheater and Wilson (1976) suggestd that
alkaline phosphatase could be used as marker for the surface
plasma membranes. It has been pointed by Simpson et al. (1981)
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b3
that 20% of the alkaline phosphatase was located on the surface
of S. mansoni, while Cesari (1974) estimated 75\ of the enzyme
from the surface membrane fraction. Similarly, Roberts et al.
(1983) observed that 34* of the alkaline phosphatase of total
worm homogenate was present in the surface membrane of S.
mansoni. These findings support our results, in which many fold
enrichment of alkaline phosphatase was observed in the isolated
surface plasma membranes, than the fresh worm homogenate.
Acid phosphatase was noticed in appreciable quantity in the
surface membranes of both the amphistoraes under study. Regarding
its function in the membranes Threadgold (1968) sug' 3ted that
the presence of acid phosphatase in plasma membranes are often
associated with transmembrane absorption. Similarly this enzyme
has also been reported from the surface membrane of S. mansoni
and from the basal apical plasma membranes of G. crumenifer and
G. explanatum (Watts et al. 1979; Dunn et a1., 1987). Khan
(1991) have reported the occurrence of ATPase enzyme in isolated
membranes of amphistomes and revealed that this enzyme occurs in + ++ + +
two forms is Na /Mg and Na /K dependent, the former form is + + +
predominant than the latter. Further Na /Mg dependent enzyme is + +
stimulated while Na /K dependent form is inhibited by ouabain. + +
Podesta & McDiarmid (1982) have also observed Mg activated and
ouabain sensitive ATPase in the surface membrane» of S. mansoni.
This shows that these parasites have the ability to make use of
the ATPase system according to their need and physicochemical
conditions of the habitat. At this stage it is difficult to
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;• I'j
suggest the nature of transport and occurrence of Na pump, which
require further kinetic studies.
Y-Glutamyl transpeptidase (Y- GTP) is a key enzyme of Y-
glutamyl cycle. It is a membrane bound enzyme responsible for the
transport of amino acids across the membrane. Y- GTP has been
demonstrated in M. benedeni but it was not found in A.
lumbricoides and F. hepatica. Singh et al (1989) reported Y- GTP
in bovine filarial nematode Setaria cervi. The results of Y- GTP
estimation in the fresh homogenate and surface plasma membrane
fractions of parasites under study revealed the higher enzyme
activity in G. explanatum followed by G. crumenifer.These results
are further supported by Abidi (1990) who have also reported the
higher activity of Y-GTP in G. explanatum than G. crumenifer.
The presence of higher concentrations of acid and alkaline
phosphatases, ATPase and Y- GTP in the surface plasma membranes
of liver parasite G. explanatum than the rumen trematode G.
crumenifer, can be explained by taking the habitat into account.
Liver parasite live in nutritionally rich habitat where simple
nutrients such as glucose and amino acid are readily availble, it
appears that absorption and transfer of nutrients are performed
by the tegument.
Quantitative differences were also observed in the
distribution of acid and alkaline phosphatases in membrane as
well as in soluble fractions of the two amphistomes under study.
It was found that acid phosphatase is much active than the
alkaline phosphatase in both the parasites. Most of the studies
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on adult trematodea have also indicated that acid phosphatase is
more active than alkaline (Von Brand, 1973; Cesari, 1974; Probert
& Lwin, 1974) .
It is evident from the results that the drugs under study
have variable effects on soluble and membrane bound enzymes of
both the amphistomes. Qualitative as well as quantitative
differences were observed in the soluble and membrane fractions
of two parasites. These differences may be attributed to the
different physicochemical nature of the habitat in which these
trematodes inhabit. Alternatinely the possible reason could be
the differences in properties of soluble and membrane bound
enzymes. Generally the differences are explained on the basis of
change in substrate specificity, optimum pH, Km and general
stability. The soluble enzymes exist in polar hydrophobic
environment of high dielectric constant. A variety of molecules
including substrates, other metabolities and ions can approach to
the soluble enzymes easily. In contrast, the membrane bound
enzymes may be largely embebded in a li.pophilic region of low
dielectric constant, with little opportunity for interaction with
smaller polar molecules. The membrane bound enzymes exist in a
relatively stable environment, the nature of which is determined
by the molecular arrangement of the membranes. Changes in
membrane composition or structure will modify the environment of
the enzymes and thus affect the regulatory system (Harrison &
Lunt, 1980).
Mebendazole has a stimulatory effect on acid, alkaline
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iZ
phosphatases and Y - GTP of soluble and membrane frations, except
acid phosphatase of membrane origin of G. explanatum while in G.
crumenifer only alkaline phosphatase was stimulated. These
results can be explained by the fact that benzimidazoles are
lipid soluble proton conductors that affect the artificial and
natural membrane system (McCraclcen & Stillwell, 1991). Therefore
mebendazole may release membrane bound enzymes and expose them to
the available drug action. The differential stimulatory effect of
the mebendazole can also be explained by taking the lipid
composition of the membrane of these parasites into
consideration. Khan (1991) has reported that liver tremato^ G.
explanatum has more lipid content than the rumen parasite G.
crumenifer in their surface plasma membranes.
Enzyme stimulation may also be the consequence of damage
and binding of the drug with tubulin moiety, resulting into
release of membrane bound emzymes. As it is well documanted that
membendazole binds selectively to parasite tubulin and therefore
tubulin is considered as the primary chemotherapeutic target
(Kohler & Bachmann, 1980,1981; Ireland et al., 1982; Coles,
1983). Effects of mebendazole to microtubule assembly can
additionally prevent the translocation of secretory products
within the tegument (Bogitsh, 1977; Bogitsh & Carter, 1977;
Borgers and Verheyen, 1976) as well as events which may be
related to the drug induced morpholigical disruption of tegument
and subtegument. It may also possible that mebendazole induces
the release of the membrane bound enzymes and become accessable
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/3
to drug molecules producing appreciable marked effects. Further,
Ahmad et al. (1987) have also reported some topographical changes
due to mebendazole in G. explanatum under in vitro conditions.
Besides these effects, mebendazole has inhibitory effect on 3
membrane bound ATPase of both the parasites and uptake of H-
glucose uptake across the tegument of G. crumenifer was also
inhibited. These results are in agreement with the previous
studies carried out on Ascaris suum and Hymenolepis diminuta
and G. explanatum (Van den Bossche, 1972; Van den Bossche and De
Nollin, 1973; McC!racken & Taylor, 1983; Ahmad & Nizami, 1991).
In cestodes the location of transport system undoubtedly be the
tegument (Pappas and Read, 1975; Pappas, 1983), while in
amphistomes Abidi (1990) and Khan (1991) also suggested that
different loci are involved in the transtegumental uptake by
using pulse chase technique of antoradiography as well as by
transport kinetics of some labelled nutrients . These authors
further suggestded that saturable carrier mediated uptake with
the involvement of diffusion component in amphistomes and follow
a saturation kinetics. Similarly cambendazole and mebendazole
also inhibit the tegumental uptake of glucose by M. expansa as
well as affecting a variety of facets of energy metabolism including reduced ATP synthesis and adenine nucleotide turnover
3 (Rahman & Bryant, 1977) Inhibition of H - glucose uptake in G.
+ ++ crumenifer can be correlated with the inhibition of Na - Mg
ATPase system of membrane origin. This further confirms that a
carrier mediated transport is involved and ATP is required. As
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'I'i
McManus and James (1975) have reported active transport of
monosaccharides by larval trematodes of Microphallus similis.
Uglem & Read (1975) have also reported the coupled movement of 2 +
deoxyglucose with Na in S.mansoni. It is further suggested
that glucose uptake by G. crumenifer occurs in part, through a +
Na coupled transport system.
Oxyclozanide is a Jcnown flukicide and unconples the oxidative
phosphorylation by blocking ATP synthesis and inhibiting MDH
activity of trematodes (Corbett & Goose, 1971; Probert et al.,
1981). Besides metabolic effects, oxyclozanide is also known to
exert a variety of neurophysiological disorders. Such effects of
oxyclozanide has also been observed in F.hepatica and G.
explanatum (Fairweather et al., 1984; Ahmad & Nizami, 1990).
Further in vitro effects of oxyclozanide on motility of G.
crumenifer reveal that this compound induces disruption in
muscular contraction and cause spastic paralysis within 3 min
(Ahmad & Nizami, 1992). This shows that this drug takes some t me 3
to produce its effect. However, in the present study of H -
glucose uptake in presence of oxyclozanide by G. crumenifer a
concentation dependent effect was observed. At a given
concentration of 24 and 66 nmoles, there was stimulation in 3
glucose uptake, whereas at 110 nmoles, a marked inhibition of H
glucose uptake was noticed. These results provide indirect
evidence that higher drug concentration causes a spastic
paralysis leading to inhibition of glucose uptake. Further,
Ahmad et al., (1987) have reported that oxyclozanide produced
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V5
roost pronounced damage at the surface of G. explanatum.
Oxyclozanide causes deformation and disappearance of surface
papillae particularly on dorsal surface where deep lesion in
which parenchymatous and internal organs were exposed. Peeling
of tegume al surface and inner lining of acetabulum was also
noticed. Deep cater like lesions were observed over the exposed
parenchymatous tissues. The topographical disorders observed may
interfere in the absorption of micromolecular nutrients.
Stimulation of acid and alkaline phosphatases of soluble fraction
of both the parasites may be the consequence of topographical
damage resulting in release of bound enzyi.js, while there was no
effect on ATPase of both the parasites. Therefore surface plasma
membrane bound enzymes seem to be destroyed by the action of
this drug, however reported effects on oxidative phosphorylation
or uncoupling reactions may be the conseqeunce of the exposed
tissue. Therefore it is likely that a single drug may have
variable effects depending on the concentration of drug. 3
Inhibition of H-glucose uptake may be a consequence of
topographical damage or metabolic disturbances due to alterations
in the enzyme molecules.
Rafoxanide has been used as flukicide. It is evident from
the results that rafoxanide has no effect on acid, alkaline and
adenosine triphosphatases of the membrane fraction of G.
explanatum while in G. crumenifer, this drug has a marked
stimulatory effect on acid and alkaline phosphatases of the
membrane fractions having no effect on ATPase. On comparing the
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vr,
effect of this drug on soluble fraction enzymes, it was found
that acid phosphatase and Y-GTP of the G. crumenifer are
stimulated and acid, alkaline phosphatases and Y-GTP of G.
explanatum are stimulated. Previous studies on F. hepatica have
shown that this drug effects intermediatry steps of metabolism
and end product formation in. main energy producing pathway. It
was pointed out that rafoxanide diminishes ATP synthesis,
resulting in increased carbon flow, followed by depletion of some
internal intermediates pool of the pathway as further metabolism
of succinate is also inhibited (Cornish & Bryant, 1976 b).
Further, Probert & Lwin (1974) have also studied the effect
of rafoxanide and nitroxynil on phosphomonoesterase of F.
hepatica. The refaxanide at 20mM was reported to have no effect
at acid or alkaline pH. 3
In presence of rafoxanide, H-glucose uptake by G.
crumenifer show a marked stimulation at various concentrations of
the drug. stimulation of acid and alkaline phosphatases of
membrane fraction in G. crumenifer may be indirectly attributed
to the transport of glucose under study, because phosphatases
have frequently been associated with absorptive functions at the
parasite surface. Cornish et al.(1977) have studied the effect of
rafoxanide on the energy metabolism of F. hepatica in vivo and
reported that glycogen and ATP levels diminished in 24 hr
treated group. The significar decrease in glycogen content
suggests that it was utilized to maintain the energy levels of
the flukes, but such effects were not or ^rved under in vitro
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7 7
conditions in F. hepatica. (Cornish & Bryant, 1976 b). This shows
that the drug has variable effects under in vitro and in vivo
conditions.
Y - GTP is an another important membrane bound enzyme which
plays a key role in the transmembranosis of amino acids across
the cell membranes. The amino acid transport involving the Y-GTP
is an example of group translocation in which amino acid binds to
the enzyme protein and the complex is transported to the inside
of cell where amino acid is released (Lehninger, 1975).
In the present study all the three drugs affect the Y-GTP
and induce stimulation in of the soluble and membrane fractions
in G. explanatum. whereas in G. crumenifer these drugs either
stimulate or had no effect, with the exception of Y-GTP of
soluble fraction, where it is inhibited significantly.
Review of the literature indicates that Y-GTP is
differentially distributed among helminths. This enzyme has been
reported from M. benedeni and body wall of Setaria ceryi
(Barrett, 1981 and Singh et al., 1989). The results of present
study further indicate that Y-GTP level is higher in liver
amphistome than rumen. Glenner et al., (1962) have reported higher
level of Y-GTP from loop of Henle and epithelial lining of the
intrahepatic bile duct. This enzyme occurs both in the soluble
and membrane bound form. It is therfore G. explanatum has higher
level of this enzyme. In the present study by and large, all the
three drugs stimulate this enzyme in order to produce glutathione
which is a reducing agent and plays a domjaant^ role in
fr :.,., ^^
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detoxification mechanism, apart from its dominant role in
transmembranosis.It is also expected that under in vivo
conditions, low reducing condition which are essential for
survival of parasites is provided by this pathway (Smyth and
Halton, 1983). This leads us to conclude that in amphistomes,
detoxification mechanism may be operative, which require further
study.
The foregoing discussion on the effect of various drug on
enzymes of soluble and membrane fractions leads us to conclude
that no generalization is possible, and the mode of action and
the degree of efficacy depends on the molecular and biochemical
configuration of membranes, binding nature of drug molecule and
physicochemical conditions of the microhabitat. Since the members
of salicylanilide group are known to induce their effect when
they are bound with the protein moiety, similarly the
benzimidazole binding with tubulin moiety are well authenticated
in the literature. However, this fact can not be ignored that
surface plasma membranes are the primary target of the drug
action, and the metabolic disorders which have been reported in
the present study as well as in previous studies may be a
consequence of secondary effects.
In order to ascertain these effects it is essential that in
vivo studies must be carried out on these lines.
Inununochemical studies:
The results of the present study clearly reveal that
oxyclozanide has a suppressive effect on the circulating
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ViJ
antibodies against membrane antigen of rumen amphistomes G.
crumenj-fer as evident by the reduction in the peaks of the
rockets drug treated animal sera.
Digenean flukes are surrounded by syncytial tegument
bounded by an exposed plasma membrane (Lumsden, 1975; Erasmus,
1977 and McLaren, 1980). The tegument is an anucleate, cytoplasmic
structure which is continuous with deeper nucleated cell bodies.
The detailed structure of the tegument is strongly indicative of
continuous turning over or shedding of the outer layers which
are replaced by material contained within the secretory bodies
arising from beneath.
The significance of the contribution of surface antigen has
been reviewed extensively by Maizels et al.,(1982) and
Butterworth (1984), they have emphasised that surface antigens
play a critical role in the expression of effective immune
responses to helminth parasites in vitro and probably also in
vivo. The active release of antigens from the surface of
helminths in yivo could have a potentially significant impact on
the immunobiology of the helminth.
Evidences are on record that immunity of the host
synergistically modulates chemotherapeutic responses of the
infective organisms (Ambroise-Thomas, 1974; Corba and Spaldonova,
1974; Grove and Warren, 1976; Tyagi & Murthy, 1986). Conversely,
chemotherapy also alters the immunologic response of the host
(Katiyar et al., 1986; Janssen, 1976). Changes in immune status
whether suppressive or stimulatory have direct bearing on
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S i]
infection/disease status.
Humoral immune response plays an important role in defense
against helminthic infections. .(Ogilvie et al., 1978). The
parasite specific immune response of the host is however expected
to be altered follwing anthelm ntic treatment. Previous work with
F. hepatica demonstrated significant decrease in antibody level
following successful treatment and has been reported to be a use
ful indicator of therapeutic success (Hillyer and Santiago de
Weil, 1979). Similar observations were made by other workers
(Grieve and Knight, 1985 and Kamath et al., 1985) in filarial
and hook worm infections.
Immunological mode of action of salicylanilide group has
been reported in some trematode parasites by a number of workers
(Hillyer and Llano de Diaz, 1976; Hillyer and Santiago de Weil,
1979 and Daugalieva, 1986). They have reported reduction in
antibody titer and complement fixation following rafoxanide and
ivermactin treatment in experimental and natural host. Our
present results are in agreement with these workers in respect to
reduction in antibody titer.
Observations of the present study show that oxyclzanide has
a suppressive effect on the antibody titer which was calculated
to be 43* suppersslon in drug treated animal. However it is not
clear how this suppression was brought about.
First possible rc asor may be that oxyclozanide possess an
inherent capacity to neutralize the already formed antibodies.
Secondly, it suppresses the antibody producing cells. Salmeron
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Hi
and Lipsky (1983) have also reported the inhibition of generation
of antibody producing cells after chloroquine treatment. At
present stage, it is not possible to suggest whether the
immunosuppression by oxyclozanide is specific or nonspecific
type, without further studies on natural amphistome infections.
It is an established fact that the effectiveness of many
drugs can be increased by the immune response of the host
therefore it is logical to try combining drug treatment with
compounds that enhance immunity. Levamisole, a known anthelmintic
is the only drug which has immunostimulating action. It has been
successfully used for chemotheraphy of mature and immature
paramphistomiasis with oxyclozanide (Schielhorn Van Veen and
Bida, 1975; Chhabra et al., 1977; Prasad, 1983) but there is no
evidence that its immunostimulating properties promote the
effectiveness of this drug.
The present work provides clear evidence that successful
therapy with a drug having no immunopotentiating action will
bring down the antibody level. The presently available drug
(oxyclozanide) for paramphistomiasis is inadequate. Therapy with
this drug leads to a state of immunosuppression in host.
Therefore there is need for newer strategy to potentiate the
activity of existing antitrematodal drug possibly by modulating
(stimulating) the host immune system while treating, as
participation of immune mechanism of host is almost obligatory
for ultimate destruction and elimination of causative organism.
Immunopharmacological agents which influence defence
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82
system, can regulate the number, functional activity and
interaction of macrophages, T and B lymphocytes. Leucocytes and
natural killer cells as well as their humoral and secretory
components. Induction of augmented immune response of host with
immunostimulating agents was initially used wi"n success in the
treatment of malignant tumour (Mathe et al., 1969) and only
recently this approach has been evaluated for the treatment of
experimental infections. Based on this concept, Adenolfi and
Bonventre (1985) were able to enhance the antileishmanial
activity of glucantine by a synthetic immunopotentiating compound
(CP - 46 - 665 - 1). Besides this, quite a number of condidate
substances are being investigated. BCG has also been studied
extensively, its immunopotentiating effect has been shown against
tumour and against a variety of protozoal infections notably
Leishmania (Smrkovsici & Larson, 1977). Glucan, a polyglucose
derivative of yeast cell wall is also a known immunopotentiating
agent used against experimental Leishmania donovani infection as
an adjuvant combined with formalin killed promastigotes (Holbrook
et al,. , 1981). Muramyl peptides, structural components of
mycobacterial cell walls, are amongst the most interesting
immunoprotentiators (Adam & Lederer, 1984).
Thus, it is essential that the use of drugs for the control
of parasite must be carefully tested in order to explore
immunopharamacological aspects of the drugs, because frequent
medication with these drugs may increase the susceptibility to
subsequent infection.
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S 3 SUMMARY
In the present study effect of some known anthelmintics on
the surface plasma membrane bound enzymes of two amphistomes i.e.
Gastrothy' x crumenifer and Gigantocotyle explanatum inhabiting
two different microhabitats have been investigated. The surface
plasma membranes of the parasites were isolated by using non -
ionic detergent, saponin. The enriched membrane pellets of the
parasites were characterized by the assay of some membrane bound
marker enzymes and trasmission electron microscopy.
Transmission electron micrograph? f both the parasite's
membranes show a typical trilarainate structural organization
similar to other plasma membranes.
Quantitative differences in the level of primary marker
enzymes were observed in the membranes of both the parasites as
compared with their respective homogenate and the former shows
many fold enrichment than the latter. The order of maximum
enrichment of various enzymes in G. explanatum is acid
phosphatase > alkaline phosphatase > Y-GTP > ATPase. While in G.
crumenifer the sequence is different i.e. acid phosphatase >
ATPase > Y-GTP and alkaline phosphatase.
On comparing the quantitative differences between G.
crumenifer and G. explanatum, all the enzyme levels were found
higher in G. explanatum than G. crumenifer. These differences may
De attributed to the physicochemical conditions of the micro -
habitat.
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Different drugs used in the present study, produced
differential effects on the membrane bound enzymes in both the
parasites, ranging from stimulation or inhibition to no effect.
Mebendazole, has no effect on the enzymes of G. crumenifer, while
ATPase of membrane origin in both the parasites is significantly
inhibited.
Rafoxanide and oxyclozanide drugs belong to the
salicylanilide group. It is interesting that the effect produced
by these drugs show some degree of similarity and dissimilarity
reflecting the variation in the physicochemical properties of the
enzyiTi molecules. Oxyclozanide had an inhibitory effect on Y-GTP
of soluble fraction in G. crumenifer while it produced either
stimulatory or no effect on ATPase, acid and alkaline
phosphatases of the two fractions in both the parasites.
In order to find out the effect of these drugs on 3
transmembranosis, H-glucose uptake was investigated in rumen
parasites in presence and absence of these drugs. Results of
glucose uptake study revealed that mebendazole has a marked 3
inhibitory effect on H-glucose uptake, which was found to be
concentration dependent i.e. inhibitory effect increases with the
increase in drug concentration. While oxyclozanide and rafoxanide 3
had stimulatory effect on H-glucose uptake by rumen parasite G.
•crumenifer.
To monitor the effect of oxyclozanide on the host immune-
system, sera were raised against the soluble fraction of whole
homogenate of G. crumenifer in drug treated and untreated
animals. The sera were tested against the partially purified
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8.J
membrane antigens by reversed rocket immuno-electrophoresis. It
was observed that height of the rocket peaks were directly
proportional to the antibody concentration. A gradual decrease in
the height of the rocket peaks corresponding to the antibody
concentration was noticed between the control and drug treated
animals. The overall suppression in antibodies level was found to ft
be 43X in drug treated animal sera. It is therefore concluded
that oxyclozanide has immunosuppressive effect.
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8f;
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V-7
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