THESIS

 

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This is to certify that the

dissertation entitled

DYNAMICS OF GLUTATHIONE REGULATION IN SCHISTOSOMA
MANSONI: CORRELATIONS WITH THE ACUTE
EFFECTS OF OLTIPRAZ

 

presented by
Dan Douglas Morrison

has been accepted towards fulfillment
of the requirements for

Ph.D. degree in Pharmacology and
Tox1cology

 

Major professor

\
Date 10/25/84

MS U i: an Afi'mnan'w Action/Equal Opportunity Institution 0-12771

 

 

 

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DYNAMICS OF GLUTATHIONE REGULATION IN SCHISTOSOMA MANSONI:

 

CORRELATIONS WITH THE ACUTE EFFECTS OF OLTIPRAZ

By

Dan Douglas Morrison

A DISSERTATION

Submitted to
Michigan State University
in partial fulfillment of the requirements

for the degree of

DOCTOR OF PHILOSOPHY

Department of Pharmacology and Tbxicology

1984

ABSTRACT

DYNAMICS OF GLUTATHIONE REGULATION IN SCHISTOSOMA MANSONI:

 

CORRELATIONS RUTH THE ACUTE EFFECTS OF OUTIPRAZ

By

Dan Douglas Morrison

Glutathione is present in adult Schistosoma mansoni (6.336 :_fl.012

 

nmoles/mg protein) at significantly lower levels than uninfected host
liver tissues (1.051 :_@.013 nmoles/mg protein) or host kidney tissue
(6.627 1. 6.613 nmoles/mg protein). Host hepatic glutathione levels
decline significantly during the course of an infection by the
parasite, while renal cortical glutathione levels are unaffected. Of
the enzymes regulating glutathione utilization, glutathione reductase
in the male parasite exhibits a specific activity of 10.3 :_4.2 mU/mg
protein, 15% that of uninfected liver. The apparent glutathione
S-transferase activity depended on substrate tested, 26 :_7 umole
conjugate fermed/min/mg protein with p— nitrobenzyl chloride as
substrate (13% of hepatic values) and 526 :- 18 pmole conjugate
formed/min/mg protein with l-chloro—Z,4-dinitrobenzene as substrate
(43% of hepatic values). Male schistosomes exhibited negligible
glutathione peroxidase activity (5 :_ 9 nmoles NADPH oxidized/min/mg
protein), while the uninfected host exhibited values of 258 :.19 nmoles
NADPH oxidized/min/mg protein fbr this enzyme. During the course of

infection, the specific activity of hepatic glutathione S-transferase

was depressed to 26% of uninfected control values (p— nitrobenzyl
chloride as substrate). Similarly, hepatic glutathione peroxidase
activity was reduced to 37% of control values. Hepatic glutathione
reductase was not significantly affected during the infection. Also,
neither of the renal enzymes, glutathione peroxidase or glutathione
reductase, were affected. Oltipraz, an antischistosomal compound,
effected a significant depletion of parasite and host glutathione
levels within 1 h of exposure _i_n y_i__v_o_ and _i_n_ _v_i_t_;_r_g at a dosage of 256
'mg/kg and 10 H5! respectively. Host tissue glutathione levels returned
to, or above, control levels by 6 h after oltipraz administration,
while parasite glutathione levels remained significantly depressed.
Uptake of [35$]cysteine or [3551cystine by schistosomes was inhibited
by oltipraz. However, the drug did not alter the relative distribution
of label once incorporated into the parasite, indicating that the
enzymes of glutathione synthesis were not directly inhibited. Oltipraz
significantly depressed surface electrical activity and the tegument
potential of male schistosomes. However, both the biochemical and
physiological effects of oltipraz (10 H!) were prevented by coincubat—
ing the parasite in 1 WE. cysteine, methionine or glutathione. The
amelioration of oltipraz-induced effects was not dependent on free
thiol groups, as neither dithiothreitol nor Zemercaptoethanol
attenuated the schistosomicidal effect of oltipraz. The inactive
analog of oltipraz (RP 36 642, 106 p_M_) did not produce effects mimick-

ing those seen with oltipraz.

ACKNOWLEDGEMENTS

I would like to acknowledge the generous support and guidance of
my advisor, Dr. James L. Bennett. Dr. Bennett's patience and under-
standing, in the genesis and development of this thesis, have helped
enable its completion despite some unexpected trials and tribulations
during my graduate career, which have given the catchwords 'in sickness

and in health' new meaning.

My special thanks to Dr. David P. Thompson for perusal of
manuscripts (ad nauseum) and for his unique style of ”guillotine“

humor .

I am indebted to Drs. 'Iheodore Brody, Ralph Fax and Paul Sato for
guidance and assistance with the developnent of the thesis proposal.
Ms. H. Cirrito and Ms. I. Mao provided invaluable technical
assistance with tissue preparations. Financial support from NIH grants

AI19889 and M14993 is gratefully acknowledged.

ii

TABIEOFCONTENTS

 

 

 

 

 

 

 

PAGE
LIST OF TABLES vi
LIST OF FIGURES vii
LIST OF ABBREVIATIONS ix
INTRODUCTION]
A. The parasite, Schistosoma mansoni l
B. Chemotherapy 2
1. General
2. Oltipraz and structure-activity relationships
3. Oltipraz spectrun of action
4. Oltipraz mechanism of action
C. Glutathione and its enzymatic regulation 6
l. Glutathione
2. Enzymatic synthesis
3. Glutathione utilization
RATIONAIE 14

 

iii

TABLE OF CCNI'ENTS (con't)

MATERIALS AND METHODS

 

A. Preparation of host and parasite tissue

 

B. Glutathione assay

C. Manipulation of schistosome glutathione levels in

 

vitro

D. High Pressure Liquid Chromatography (HPDC) -—--—--

 

E. Enzyme assays

F. Mechanical, surface electrial and tegumental

 

potential measurements

Chapter 1: Best—parasite differences in the regulation

of glutathione utilization

 

Introduction

Results

 

 

Discussion

Chapter 2: Acute effects of oltipraz and its antagonism

.ig vitro

 

Introduction

 

Results

iv

15
l6

l6
l7

17

19

20
21

24

29
29

TABLE OF CCNI'ENTS (con't)

 

Discussion 38

Chapter 3: Correlation of schistosomal glutathione

levels with physiological parameters

 

 

 

Introduction 46
Results 46
Discussion 49

Chapter 4: Effects of schistosomal infection on host

regulation of glutathione levels

 

 

 

 

Introduction 55
Results 56
Discussion 59
SUMMARY AND CONCLUSIONS 6%
BIBLIOGRAPHY 62

 

Table

mansoni and uninfected mouse liver tissue

LIST OF TABLES

Drugs used in the treatment of human schistosomiasis

Glutathione S-transferase activities of Schistosoma

 

 

Mouse hepatic glutathione-related enzyme activity:

 

Effect of Schistosoma mansoni infection

 

‘vi

Page

23

58

Figure

10

ll

12

13

LIST OF FIGURES

 

Chemical structure of oltipraz

 

Chemical structure of glutathione

 

Y-glutamyl cycle
Chemical structure of buthionine sulfOximine

Dose—dependent depletion of schistosomal glutathione

 

by’diamide
Oltipraz effect on glutathione content in infected

mouse hepatic and renal cortical tissue and in male

 

schistosomes

Dose-dependent depletion of schistosomal glutathione

 

by oltipraz

Effect of oltipraz and various compounds on

 

schistosome glutathione levels_in vitro
Oltipraz effect on schistosomal glutathione levels
_in vitro: Aerobic vs anaerobic environment

[358]cysteine uptake and incorporation into

 

schistosomes in vitro

 

[14C]glucose uptake into schistosomes in vitro

Surface electrical activity of male schistosomes in

 

vivo: Oltipraz effect
Tegument potential of male schistosomes in_vitro:

Oltipraz effect and antagonism by cysteine

'vii

Page

11

22

30

31

32

34

35
36

37

39

Figure

14

15

l6

17

18
19

20

LIST or FIGURES (con't)

Hypothetical reaction sequence to explain oxygen-

dependency of oltipraz-induced depletion of

 

schistosomal GSH in vitro

Dose-dependent depletion of schistosomal glutathione

 

by BCNU

Tegument potential of male schistosomes in vitro:

 

Effect of BCNU and antagonism by cysteine

Dose-dependent depletion of schistosomal glutathione

 

by 880
Oltipraz effect on schistosome fecundity in vitro —-

Effect of various reagents on oltipraz inhibition of

 

schistosome fecundity in vitro
Glutathione levels in host hepatic and renal cortical

tissues during the course of Schistosoma mansoni

 

 

infection

viii

Page

42

47

48

50
51

52

57

AT-125

BSO

GRed

GSH

GTP

HPLC

HS/RPMI

NAD(P)H

SCE.

LIST OF ABBREVIATIONS

Irwas,SS)-a-Amino-3-chloro—4,5—dihydro—S-isoxazoleacetic
acid

1,3—bis [2-chloroethyl]-l-nitrosourea
L—buthionine-S-R—sulfoximine

Dithiothreitol

Glutathione peroxidase (EC 1.11.1.9)

Glutathione reductase (EC 1.6.4.2)

Glutathione, reduced fOrm (Y-L—glutamyl-L—cysteinylglycine)
Glutathione transpeptidase (EC 2.3.2.2)

Glutathione S—transferase (EC 2.4.1.18)

High pressure liquid chromatography

1:1 mixture of horse serum (heat-inactivated) and RPMI 1640,
to which penicillin (100 U/ml), streptomycin (100 ug/ml) and
Fungizone (0.5 pg/ml) were added

Nicotinamide—adenine dinucleotide (phosphate)

Standard error of the mean

Trichloroacetic acid

ix

INTRODUCTTON

A. The parasite, Schistosoma mansoni:

 

Schistosoma mansoni is a digenetic parasite, which debilitates

 

millions of people in tropical regions throughout the world. The
regions of endemic schistosomiasis are restricted to tropical climates
because of the presence of the snail vector in which the schistosome
develops during one stage of its two-host life cycle. '§;_ mansoni
adults, residing in the mesentaric venules lining the intestines of the
human, oviposit approximately 300 eggs/day/worm pair [99]. Up to 70%
of these are dislodged from the venule into the blood stream and become
entrapped within the hepatic sinusoids of the liver or the
microfenestrations of the spleen, resulting in the hepatosplenic
pathology which characterizes long-term, heavy worm burden infections
[130,131,132]. The overt disease symptoms, hepatomegaly, splenomegaly,
portal hypertension and esophageal varices, can be prevented, even in
mice heavily infected with schistosomes by inhibition of oviposition.
Also, the disease state shows signs of amelioration, by gradual host
repair of damaged tissues, if egg production is halted after the onset

of hepatosplenic disease [129].

An increase in the prevalence and intensity of schistosomiasis is
occurring in the tropics because agricultural irrigation is rapidly
expanding in countries where the disease is endemic, providing a larger
habitat fer the snail host. To interrupt transmission of the disease
requires effective sanitary waste disposal, large—scale snail control,
or mass chemotherapy. Hewever, field-trials have indicated that the
best results are obtained with chemotherapy [78], and chemotherapy will
likely remain the only viable approach in the control of the disease
for the extended future. HOwever, due to the proclivity of the human
population to become reinfected, mass chemotherapy is an expensive

temporary solution.

B. Chemotherapy:

1. General: Several compounds are available which exhibit
antischistosomal activity (Table 1, from [16]), however, the use of the
antimonials, niridazole and hycanthone is limited due to host toxicity.
Metrifonate is not effective in eliminating mansoni schistosomiasis,
which limits its use. Research is warranted on the other
antischistosomal compounds to attempt to determine their modes of
action in efforts to design more efficacious drugs or cheaper
substitutes that still retain schistosomicidal activity. Further
research is also warranted to forestall or circunvent possible problems
of resistance that may develop to drugs currently available and in use,
or the possible discovery of untoward effects of the newer drugs on the
host. Ideally, the antiparasitic would be effective in a single oral

dose, of low cost, and effective against all stages of the parasite

 

 

Table 1: Drugs Used in the Treatment of Human Schistosomiasis
Antischistosomal Antischistosomal Cbmments
compoung Spectrum of
(date) Action
Antimonials __._ _._, __._ j_._, _S__._ __:_ Limited use due
(1918) to toxicity
Metrifonate _;__;_ Minor side
(1962) effects
Niridazole EL.EEJ.§_._; Cbnvulsant,
(1965) carcinogenic
Hycanthone §;.EkJ _J__;_ Hepatic necrosis,
(1967) carcinogenic
Oxamniquine §;.Eh. Minor side
(1973) effects
Amoscanate §;_ . Hepatotoxicity
(1978) _S_:__r_n_._ (?) ,_S_.__h_._ (7)
Praziquantel _S_._ m., .5; ., _S__.__h__
(1979)
Oltipraz §.:. _I_n_:_, _S_._ ___:_
(1979)

 

a Date of first reported use in humans with schistosomiasis.
Schistosoma mansoni (S. m.), Schistosoma haematobium (S. h.)

 

 

and Sohi§tosoma japonicum (S. j.).

 

[67] .

2. Oltipraz structure and structure-activity relationships:

 

 

 

Oltipraz (35 972 R.P.; 4-methy1—5-[2-pyrizinyl]-l,2—dithiol-3-thione;
Figure l) was synthesized and patented in the mid-1970's by Rhone
Poulenc Sante, Paris [9]. Congeners of this drug exhibit amebicidal
[9] and fungicidal [12] activity, while some other compounds with the
1,2-dithiole-3-thione ring structure exhibit diuretic activity [38].
The structure-activity relationship for oltipraz is rather stringent
[25]. The presence of the thione and an adjacent aromatic ring con-
taining two nitrogens are structural properties required for
antischistosomal activity [23]. Further, while oltipraz is extensively
metabolized [19], none of the metabolites which have been identified

possess any antischistosomal activity [77].

3. Oltipraz spectrum _o__f action: In man, oltipraz is effective in

 

erradicating _S_.mansoni, _S_.haematobium and _S_.intercalatum infections

 

 

[49,108] , but the drug is ineffective against _S_. jamnicum [24].
Oltipraz is less active against schistosomula than against mature adult

schistosomes [89] .

4. Oltipraz mechanism 313' action: Oltipraz is a slow-acting drug,

 

two or more months may pass before the worm burden is totally
erradicated with a curative dose. In mice given very high doses of the
drug, evidence of worm lethality is observed after 48 h, with all
schistosomes being killed by 14 days post-treatment [89] . The

mechanism of action which leads eventually to the schistosomacidal

Figure 1: Chemical structure of oltipraz.

OLTIPRAZ

outcome remains unknown. There are no published reports on acute
effects of this drug, nor have reports been published 0“.12”21E£9
studies to determine the direct action of this compound on the parasite
which are independent of the variables of the host response to drug
action. With oltipraz, and various structural analogs of oltipraz, a
positive correlation exists between antischistosomal activity and the
depression of glutathione levels in the parasite [25]. Although not
studied acutely, these investigators also observed a concomittant
elevation in the glutathione levels of various host tissues
post-treatment, suggesting that a biochemical difference exists between
host and parasite in the regulation of this important intracellular

thiol compound.

C. Glutathione and enzymatic regulation of glutathione:

l. Glutathione: Glutathione (Y-L—glutamyl-L-cysteinylglycine;

 

GSH) is a tripeptide of molecular weight 307.33, shown in Figure 2.
The pKa of the sulfhydryl is 9.2, therefbre GSH carries a net negative
charge at physiological pH (as does cysteine, with a pKa of the
sulfhydryl at 8.18). GSH oxidizes spontaneously at physiological pH to
form the disulfide, oxidized GSH. Alternatively, GSH can react with
cysteine or protein sulfhydryls to fbrm mixed disulfides. The role of
GSH in the overall regulation of enzyme function is an area of much
current study. "Essential" protein sulfhydryl groups are ubiquitous,
whether in a catalytic role or in determining protein confbrmation, as
is indicated by the inactivation of a large nunber of these enzymes by

oxidants such as diamide, N—ethylmaleimide or 5,5'-dithiobis

Figure 2: Chemical structure of glutathione

CIOOH

HzN-CIZ-H
CH2
CH2

HN

HS- cuz-C:H
c=o
~+
CH2
c'oow

GLUTATHIONE

1-L-glutamyl - L-cystoinylglycine

(2-nitrobenzoic acid). For this reason, reducing agents, such as
2-mercaptoethanol or dithiothreitol are commonly used to stabilize
enzymatic function. In light of the effects of thiol/disulfide
exchange on enzyme activity, this covalent modification may play an
important role in the biological control of various metabolic pathways.
Further, the thiol/disulfide status of GSH may modulate enzyme activity
[50], and recently, GSH disulfide has been shown to inactivate,
destabilize, and enhance proteolytic V susceptibility of
fructose-1,6-bis-phosphate aldolase [104]. The importance of GSH is
underscored by its ubiquitous occurrence in every organism examined.
GSH is a cofactor, coenzyme or substrate for an encyclopedic number of
enzymatic reactions. On the molecular level, the most conspicuous
aspect of cellular GSH depletion is its relationship to cell damage.
That GSH provides an important cellular defense against oxidant injury
has been well documented [16,68,133]. During a state of GSH
deficiency, reactive metabolites, formed .12 ‘sitg, bind to
macromolecules of biologic importance and cellular toxicity results
[50]. The formation of oxygen-dependent free radicals and peroxides

result in the peroxidation of membrane polyunsaturated fatty acids.

2. Enzymatic synthesis: The synthesis of GSH occurs in a series

 

of 6 reactions of the Y-glutamyl cycle from precursor amino acids
(Figure 3, from [92]). The enzymes of this cycle have been reviewed
[94,96]. The availability of the precusor amino acid, cysteine, is the
rate-limiting factor in GSH synthesis. The physiologic concentration
of cysteine is about an order of magnitude lower than the Km of

Y-glutamylcysteine synthetase for cysteine [114]. According to the

Figure 3: Y-glutamyl cycle

R-CH-COOH
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report of Beatty and Read [l4], cysteine or cystine supported GSH
synthesis in isolated rat hepatocytes better than methionine. However,
the determination of GSH synthesis by these authors, which was by the
rate of accumulation of cysteine-glutathione mixed disulfide in the
incubation media, probably resulted in erroneous estimates of GSH
synthesis, due to inhibition of GSH efflum from isolated rat
hepatocytes by methionine [7]. Also, methionine, a precursor of
cysteine via the cystathionine pathway, has been shown to be a good
precursor fOr rapid GSH synthesis in hepatocytes [13,113]. The kidney
differs from the liver, in that cysteine, as cystine, is readily taken
up by tubular epithelium, supporting the requirements for the rapid
turnover of glutathione in kidney [98,106], whereas methionine is
poorly supportive of rapid GSH synthesis, probably because of a
deficiency of cystathionase [98]. GSH regulates its own synthesis by
non-allosteric competitive inhibition of the Y-glutamate binding site
of Y—glutamylcysteine synthetase [39,114]. The y-glutamyl bond in the
tripeptide renders GSH resistant to degradation by «a-peptidases,
however, this bond is susceptible to hydrolytic and transpeptidation
reactions leading to products recoverable by the cell. GSH synthesis
can be inhibited by buthionine sulfoximine (Figure 4), a potent and
specific inhibitor of Y-glutamylcysteine synthetase [54,55,56,58].
L-2-imidazolidone—4-carboxylate inhibits the enzyme S—oxoprolinase
[58]. Potent, irreversible inhibition of Y-glutamyl transpeptidase
activity can be attained with AThlZS [61]. The structure-activity
relationships of substrate analogs on the inhibition of the Y-glutamyl

cycle enzymes have been studied [57].

11

Figure 4: Chemical structure of buthionine sulfoximine.

C00-

4- I
H3N-C'I-H
«I»... I:
0=lSl-CH2-C': -H
NH CH2

CH3

BUTHIONINE SULFOXIMINE

12

3. Glutathione utilization: Intracellular GSH is converted to

 

oxidized GSH by selenium—containing glutathione peroxidase (GPx), which
also catalyzes the reduction of H202 and other peroxides. GPx is
specific for its hydrogen donor, GSH, and nonspecific for the
hydroperoxide [95]. Therefore, GPx catalytically detoxicates a range
of substrates from H202 to organic hydroperoxides. Although GPx shares
the substrate H202 with catalase, it alone can react effectively' with
organic hydroperoxides as well. Selenium—independent GPx activity
describes the peroxidase activity of glutathione transferase [88]. In
male weanling rats maintained on a selenium-deficient diet, GPX
activity fell to undetectable levels. When these animals were also

deprived of vitamin E and sulfur-containing amino acids, a fatal

hepatic necrosis deve10ped [63,116].

The reduction of oxidized GSH, with concomitant oxidation of
NADPH, is catalyzed by the flav0protein glutathione reductase (GRed) in
an essentially irreversible reaction, accounting for the very high
GSH:oxidized GSH ratios found in cells [95]. The importance of this
enzyme is most apparent in cells which no longer synthesize
macroomolecules, such as the mature erythrocyte where maintainance of
adequate levels of reduced GSH is essential fOr the deformability and
flexibility of these cells [71]. In rat hepatocytes, the protective
role of GRed against adriamycin-mediated toxicity was demonstrated by
the inactivation of this enzyme with BCNU [8]. In these experiments,
the rapid recycling of oxidized GSH to GSH by GRed ameliorated the
lipid peroxidation seen when GRed was inactivated. Glutathione

S—transferase performs the catalytic function of conjugating

13

potentially harmful electrOphilic compounds of hydrophobic character
‘with the nucleOphilic GSH to produce a compound less toxic and more
water soluble than the parent compound. By reason of their high
affinity and concentration, they may also act as scavengers for
alkylating agents. In a suicide—manner, the covalent bond fbrmation
between the transferase and the highly reactive electrophile
detoxicates the compound, at the expense of further enzyme function

[73].

RATIONALE

The overall research objective for studying biochemical differ-
ences between the host and parasite is the possibility that this
approach could lead to the rational development of new families of
drugs which exhibit antiparasitic activity at levels of drug which are
not toxic to the host. The rationale fbr investigating the mechanism
of action of oltipraz is to determine the biochemical differences
between these organisms which result in schistosomicidal activity with
only minor side effects noted in the host. This may, in turn, lead to
the developnent of better, safer and less expensive new

chemotherapeutics .

14

MATERIALSANDMEIHODS

A. Preparation of host and parasite tissue: Adult Schistosoma

 

 

 

mansoni, 45-55 days post-infection were dissected from the hepatic and
mesentaric veins of Swiss Webster female mice by the method of Fetterer
_e_§ _a_l_. [45]. Where male parasites were used, worms were dissected
into media containing 0.05% sodiun pentobarbital, mechanically
separated and the males placed into fresh media and maintained at 37°C

until assay.

For _i_r_1_ vivo experiments, mice were dosed by gavage at either 125
mg/kg or 250 mg/kg oltipraz in peanut oil or peanut oil vehicle and

sacrificed at various times post dosing.

For anaerobic experiments, 40 male parasites were incubated 18
hours at 37 °C in 25 ml PIS/RPMI. Prior to incubation and inmediately
after addition of worms to the media, nitrogen gas was passed through
an oxygen trap (0.2 fl pyrogallol in 0.2 E NaOI-I) , then bubbled through

the media to replace dissolved oxygen.

For egg-laying experiments, fifteen pairs of animals were
transferred at random to 50 m1 of media in 250 ml Erlenmeyer flasks.
The medium was a 1:1 mixture of RPMI 1640 and heat inactivated horse
seruu, to which had been added 100 U/ml penicillin and 100 ug/ml
streptomycin (media components from Gibco Laboratories, Grand Island,
NY). Mercaptoethanol was added to a final concentration of 50 pg.

Drug or vehicle were added to the media in a volune not exceeding 100

15

16

ul. Flasks were then incubated for 72 h at 37°C, with continuous

mechanical agitation.

B. Glutathione assay: At the end of the incubation period, worms

 

were filtered, weighed and homogenized (1:10, w/v) in 6%
trichloroacetic acid (TCA). Host tissues were perfused ‘with ice—cold
1.15% KCl to clear blood from the organ and then homogenized 1:20 (wzv)
in 6% TCA. Aliquots were removed fOr protein determination by the
method of Albro [2]. Following 1000 x gcentrifugation, GSH was
determined by a modification of the method of Ellman [44], where to 100
pl aliquots of supernatant were added 800 pl of 0.3 g NaZHPO4, pH 8.2
and 100 pl 0.04% l—fluoro—2,4 dinitrophenol. Absorbance was recorded

at 412 nm for freshly prepared standards and samples.

 

 

C. Manipulation of schistosome GSH levels in vitro: Schistosome
GSH levels were altered by treatment of male parasites with diamide
[88]. Tb determine the extent of lipid peroxidation, malondialdehyde,
which is fbrmed as a breakdown product of polyunsaturated fatty acids

was measured by the thiobarbituric acid assay of Buege and Aust [29].

Parasite GSH levels were measured after incubation in varying
concentrations of BCNU (1,3-bis [Z-chloroethyl]-l-nitrosourea) for 3 h
or 880 (L-buthionine—S-R-sulfOximine) fbr 6 h. The dose-dependency of
oltipraz in depleting parasite GSH levels was established fbr l h, and
the antagonism of oltipraz depression of schistosomal GSH by various

compounds was evaluated at 18, 36 and 72 h.

17

D. High pressure liquid chromatography (HPLC): Male schistosomes
were transferred to wells (multi—well tissue culture plates, Flow
Laboratories, Inc., McLean, VA) containing 2.5 ml RPMI 1640, 3 x 105
dpm/well [3581cysteine or [3581cystine or [14C]glucose (Amersham Corp.,
Arlington Heights, IL) and other excgenously added components as
indicated. Plates were maintained at 37 C in a dark chamber, for the
time periods specified. Parasites were weighed and homogenized 1:10
(wzv) in 3.5% perchloric acid. The homogenate was centrifuged 12000 x
g, 10 min. One-half ml aliquots of acid-soluble supernatant were
derivatized and the derivatives analyzed by HPLC as described by Reed
.g£.§l. [114]. Eluant was monitored at 352 nm and collected in 0.5 m1
fractions. Five ml ACS (Amersham Corp., Arlington Heights, IL) was

added to each fraction and fractions were assayed for radioactivity by

liquid scintillation (Beckman Instruments, Inc., Fullerton, CA).

E. .Enzymg’ .assays: The enzyme activity of Y-glutamyl
transpeptidase (Y-GTP) was determined according to a modification of
the method of Orlowski and Meister [108]. Tissue homogenates (1:20,
w:v) were prepared in 0.1 fl KZHPO4, 1 mg GSH, pH 8.0. A 20 pl aliquot
of the supernatant fraction (25,000 x g, 20 min) was added to 480 pl
substrate buffer consisting of 5.55 m! L-Y—glutamyl—p—nitroanilide,
11.1 m]! litJCl2 and 110 mg Tris-HCl. E‘nzymic activity was monitored at
pH's from 6.0 to 9.5 in increments of 0.5 pH unit. The reaction was
stopped with 1 ml 1.5 g acetic acid and the p—nitroaniline product
formed was measured at 410 nm against a reference solution. Enzyme

activity is expressed as uncles p-nitroaniline formed/min/ mg protein.

18

Glutathione S—transferase (GTr) activity was examined in tissue
cytosol fractions, prepared as above, in the presence of 1m}! (SH, with
1,2-dichloro-4-nitrobenzene or p-nitrobenzyl chloride as substrate.
The initial velocities of glutathione conjugate formation were measured
spectrophotometrically at ambient temperature according to the
procedure of Habig et 31. [64]. Specific activity is expressed as
nmoles product/min/mg protein, after correction for spontaneous

nonenzymatic conjugation.

Glutathione reductase (GRed) activity was assayed by the method of
Beutler [l7]. Either 50 pl of 2 mg NADPH (or NADH) was used as
substrate and the rate of change in optical density at 340 nm was
recorded. The oxidation of 1 mole NADPH (or NADH) per minute was used

as a unit of GRed activity.

Glutathione peroxidase (GPx) was measured by both direct and
indirect ° assay methods. Both assays measure seleniun— and
nonselenium—dependent GPx. The direct assay was performed by the
method of Mills [100] as modified by Hafemangtal. [65]. Parasite
tissues were prepared as described for the Y—GTP assay (rather than 4
volunes 0.15 _t_4_ KCl used by Hafeman _e_t_:_ El) . The indirect assay for the
measurement of GP): was by the method of Beutler [18] . The rate of for-
mation of oxidized GSH was measured by the change in absorbance upon

the oxidation of NADPH to NADP+.

19

F. Mechanical, surface electrical and tegumental mtential

 

measurements: Recordings of longitudinal muscle tension and muscle

 

activity were made from male schistosomes as previously described [45] .
Surface electrical activity was recorded by the use of suction
electrodes as described by Semeyn gt _a__l_., [122]. Recordings of tegu-
mental potentials were obtained by using glass microelectrodes as

described by Thompson _e_t_: 511. [129].

CHAPTER ONE: HOST-PARASITE DIFFERENCES IN THE REGULATION OF GLUTATHIONE

Introduction:

 

Schistosome glutathione (GSH) levels declined after .in vivo

 

treatment with the antischistosomal drug, oltipraz, while the GSH
content of various host tissues was observed to increase [26],
suggesting the possibility that there may be chemotherapeutically
vulnerable sites in the parasite relating to biochemical differences in
GSH metabolism. The relationship of GSH levels to the maintenance of
schistosome viability has not been examined, although GSH has been
detected in relatively high concentrations in almost all cells of
living organisms, including the schistosomes. Presence of the enzymes
of the Y—glutamyl cycle which regulate GSH synthesis have been reported

only for the cestode Moniezia benedeni [90]. Because of its reactive

 

sulfhydryl group, GSH is involved in a large variety of chemical
reactions. The GSH S—transferase family of enzymes utilize GSH to
protect proteins and membranes against reactive electrophilic species
by the fOrmation of GSH adducts. GSH serves to protect cells against
peroxides formed during oxidative metabolic processes in a GSH
peroxidase catalyzed reaction [4]. Further, in addition to its role as
cofactor fOr numerous reactions of cellular metabolism [48,81], GSH
regulates the redox status of protein thiol groups, thus modulating
various metabolic processes and membrane events [51,106], underscoring
the importance of this tripeptide to both host and parasite. The
enzymatic regulation of GSH by §;. mansoni was investigated in this

report.

20

21

Results:

Diamide effected a dose-dependent depletion of schistosome
intracellular GSH (Figure 5). The production of'malondialdehyde,
however, was not observed after 1 h incubations of parasites in diamide
at concentrations as high as 3 my, even though the parasites were

moribund at this concentration.

Male §;_ mansoni possess low levels of the enzyme Y-glutamyl
transpeptidase. The parasite enzyme activity (68 nmoles product
formed/min/mg protein) is of the same order as uninfected mouse liver
tissue (59 pmoles product fOrmed/min/mg protein). Uninfected mouse
kidney cortex preparations exhibited greater activity, 331 umoles
product fOrmed/min/mg protein. The pH optimum fOr parasite enzyme

activity, pH 8.5, is the same as that observed fer the host tissues.

Male parasites possess GSH S-transferase activity, with an optimum
for catalytic activity at pH 8.0. The activity of the enzyme occurs
with a.slight1y higher Km.than host liver, but the reduced affinity for
substrate is not due to inhibitory factors in the parasite cytosol, as
the cytosolic fraction did not inhibit GTr kinetics (purified GTr from
equine liver, Sigma), although specific activity did decline over time,
suggesting the presence of proteases in the parasite supernatant. The
GTr activities of male and female parasites and of uninfected mouse

liver are shown in Table 2.

22

Figure 5: Dose-dependent depletion of schistosome glutathione by
diamide. Twenty adult male schistosomes were incubated in 2.5 ml RPMI
1640 containing various concentrations of diamide, as indicated.
Incubations were performed at 37°C, in a dark chamber for 1 h. values
represent two or three separate experiments with triplicate groups of
20 animals per group. vertical lines are‘: l S. E., Asterisk = p <

0.05.

 

 

 

 

.x. u
L .x. ..
O
R
T.
N
0
C 1..
3i 2w 1.. O
O O O O

T- 2.39:. a: . $.5sz ~20. 5330

3.0

|.O

.03

DIAMIDE (MM)

23

Table 2: Glutathione S-transferase activities of Schistosoma mansoni
and uninfected mouse liver tissue.

Substrate

péNitrobenzyl Chloride l-Chloro—2,4-Dinitrobenzene

 

 

 

Liver 199126 12101200
§;_mansoni male 26: 7 526:169
§;_mansoni female 9: 4 * 172:141 *

Glutathione S—transferase activity is expressed as nmoles product/min/
lug protein, after correction fOr spontaneous nonenzymatic conjugation.

Male _S_._ mansoni possess 10.3 i 0.2 n'U/mg protein GRed activity,
which is 15.1% of mouse liver enzyme activity with NADPH as cofactor.
‘With NADH, GRed activity was reduced 90% in both parasite and mouse
liver tissue. The presence of inhibitory factors in the schistosome
homogenate was considered unlikely, as boiled worm homogenate did not

inhibit the activity of purified yeast GRed (obtained from Sigma).

Male schistosome GPx activity was not significantly above blank

values with either the direct or indirect assay.

Discussion:

 

In this study, reduction of parasite GSH by diamide treatment .in
.XiEEQ. to 30% of control did not yield a measurable increase in
malondialdehyde equivalents. GSH depletion to a critical level, about
20% of control, was required before an increase in lipid peroxidation
was observed in rat hepatocytes [136]. As the parasites were dead or
dying at diamide concentrations effecting 60—70% GSH depletion, it
would appear that diamide toxicity may not be dependent on membrane
damage due to lipid fragmentation occurring as a result of peroxide
attack. The toxicity observed fbr diamide may be attributed to
nonspecific sulfhydryl oxidation [67]. Further, membrane proteins
begin to fOrm intrachain disulfides in cells in which GSH has been
depleted by 60-70% [89], resulting in altered membrane properties
[112,124].

25

The values obtained fOr Y-glutamyl transpeptidase activity from
parasite supernatants may be misleading, since no ultrastructural
studies on the localization of the enzyme were perfbrmed and the
specific activity reported is fer soluble protein from the entire
animal. In general, higher enzyme activity is located on the external
surface of cells which exhibit prolific secretory or absorptive
functions. Therefbre the enzyme may be localized to the tegument of
the schistosome, which comprises only 1-2% of the total worm protein
(unpublished results). As tissues are essentially impermeable to
exogenous GSH without the transpeptidase [66], these results suggest
that schistosomes may be capable of absorbing GSH from the host blood
stream, possibly coupled with amino acid uptake by the classical

Y-glutamyl cycle [97,99].

Schistosomes are capable of performing phase 2 conjugation
reactions, suggesting that GTr activity'may play a role in detoxication
of chemotherapeutic agents. Female parasite activity at 30-40% of male
GTr values is without explanation, however, other sex-related
differences in the activities of schistosomal enzymes have been
observed [37]. Further, the large amount of soluble protein released
from the female digestive system during homogenization. may' have
resulted in lower specific activities than are actually present

intracellularly.

26

That schistosomal GRed activity was only 15% as active as
mammalian liver tissue could indicate that the parasite is more at risk
to oxidative damage when GSH levels are depleted. However, in light of
the fact that §;_ mansoni are functional anaerobes [27], the low level
of GRed activity is not surprising. The level of GRed would still be
the primary determinant of the availability of reduced glutathione
because GRed is considered to be the rate limiting factor in the redox

cycling of GSH [104,125].

The results with the two pyridine nucleotide substrates on host
and schistosomal GRed activity suggest that the enzyme is NADPH
specific and that there may be one or more transhydrogenases present in
the crude homogenate capable of transferring the reducing equivalent
from NADH to NADP+. The importance of functional mitochondria to the
maintenance of control levels of reduced GSH in rat tissue was shown by
Jocelyn [78], where although the reduction of GSH by diamide was
mediated by GSH oxidation, GSH levels were maintained due to
regeneration by NADH driven transhydrogenase and NADP+-specific GRed.
The functional status of schistosome mitochondria has not been clearly
elucidated. The results obtained in the present study showing no lipid
peroxidation in the presence of diamide suggest that the activities of
parasite GRed and transhydrogenase reactions are adequate to the task
of rapidly recycling GSH from oxidized GSH in the face of an oxidant

Stress o

27

That the lack of significant GPx activity in the male schistosome
is compatible with parasite viability indicates that under physiologic
conditions either the parasites are not exposed to peroxide stress or
have evolved other mechanisms to minimize or repair peroxide-induced
toxicity. Circulating concentrations of lipid peroxides in the blood
stream are negligible, likely due to the high GPx activity reported fbr
host red cells [17]. Endogenous peroxide formation is probably minimal
in schistosomes as reports on parasite metabolism indicate a reliance
on reductive or conjugative reactions fOr drug metabolism
[41,42,43,l03]. Also, schistosomes do not possess a functional
cytochrome mixed function oxidase system [10], further minimizing the
level of toxic insult to the parasite due to GPx deficiency.
Furthermore, there is a nonenzymic decomposition of peroxide by GSH,
‘proportional to GSH levels [100], which may provide adequate protection
for the parasite from low levels of intracellularly generated

peroxides.

A. chemotherapeutic possibility, the inhibition of ‘v-glutamyl
transpeptidase would prevent the normal extracellular breakdown of GSH
and inhibit translocation of Y-glutamyl amino acids intracellularly.
In mice treated with AT 125, an irreversible Y-glutamyl transpeptidase
inhibitor, and in two human patients exhibiting congenital Y-GTP
deficiency, glutathionemia and glutathionuria developed, with marked
enhancement in the renal excretion of Y-glutamylcysteine, cysteine and
cystine also being observed [60,63]. The toxicity of irreversible
inhibitors of Y-GTP to the schistosome have yet to be studied.

Therefore, while enzymatic differences between host and parasite were

28

observed in this report, the possibility for the rational design of a
selective chemotherapeutic compound based on these differences is

marg inal .

CHAPTER TWO: ACUTE EFFECTS OF OUTIPRAZ AND ITS ANTAGONISMIINTVITRO

Introduction:

 

Bueding e_t_ 31. [26] observed a depression of parasite glutathione
(GSH) content as one of the earliest biochemical changes after adminis-
tration of oltipraz _i_n 2.13.9: As the reduction in parasite GSH was
accompanied by an increase in host tissue levels of (BH, the possibil-
ity for the rational design of a drug selectively toxic to the parasite
based on interference with parasite glutathione metabolism or synthesis
suggests itself. Tb further define the relationship between the
antischistosomal activity of oltipraz and parasite (SH levels, the
acute effects of oltipraz on various physiological parameters of S:-

mansoni 3:9. vivo and _i_n_ vitro are presented.

Results :

_I_r_1__y_i_v_o_, oltipraz (250 ng/kg) , effected a significant reduction in
male parasite, host liver and host renal cortex GSH levels by 1 h, with
maximun depression at l h for host tissues and 3 h for parasite tissue
(Figure 6). (EH levels of the host rebound to or above control levels
at 6 h. However, parasite GSH levels remained significantly depressed
at that time. in y_i_t_r_g, oltipraz (10 pg) significantly depressed
schistosome GSH levels by l h, (Figure 7) an effect not observed when
parasites were coincubated in GSH (1 my.) , cysteine (1 my) or methionine
(1 It!) . Neither UI'I‘ nor 2-mercaptoethanol (100 p]! or 1 ml!) blocked the

oltipraz-induced depression of schistosome GSH in vitro (Figure 8).

29

 

30

Figure 6: Oltipraz effect on glutathione content in infected mouse
hepatic and renal cortical tissue and in male schistosomes _ig 23119.
Infected mice were dosed with 250 mg/kg oltipraz in peanut oil by
gavage. Mice were allowed food and water ad _l__i_b. Worms from 6 mice
were pooled and GSH was assayed from groups of approx. 40 males [0].
Tissue slices of host liver [I] or kidney cortex slices [9] were
pooled and 5 groups of approx. 100 mg (wet weight) tissue were assayed

for GSH: Asterisk = p<0.05 from time 0 levels.

 

 

 

 

 

A7520... 9.: . 3.0:. 5 .30

Hours

31

Figure 7: Dose-dependent depletion of schistosomal glutathione by
oltipraz. Twenty adult male schistosomes were incubated in 2.5 ml RPMI
containing various concentrations of oltipraz, as indicated.
Incubations were performed at 37°C, in a dark chamber, for 1 h. Values
represent two or three separate experiments with triplicate groups of

20 animals per group. Vertical lines are i l S.E., Asterisk = p <0.05.

*lO'

C ONTROL
\

*IU'

 

 

30

 

 

q I a
O. W O O

b-2585 a: 3825 50.1.2540

OLTIPRAZ (pM)

32

Figure 8: Effect of oltipraz and various compounds on schistosome
glutathione levels in 17.3.29: Paired schistosomes were incubated in 50
m1 HS/RPMI at 37 °C, in a dark chamber with continuous mechanical agita-
tion. [as] = p < 0.05 from control value at the same time point
(without oltipraz control). [*1 = p < 0.05 from oltipraz value at the
same time point. + oltipraz is schistosomes incubated in 1 [1M
oltipraz. [c] is glutathione, reduced form; [I] is cysteine (1 m_M_);
[c] is methionine (1 mg); [fir] is dithiothreitol (100 u!) and [*1 is
oltipraz (1 u!) . Values represent means of triplicate samples of males
which were separated from female parasites at the end of the experi-

ment .

GSH (nmoles-mg protein" )

.40‘

as
9

to
9

 

With Oltipraz Without Oltipraz

 

 

O
0
3
z}
‘4
I l I _"—"
18 36 72 72

TIME (Hrs)

33

Surprisingly, oltipraz (l u!) lowered parasite GSH levels only in an
aerobic environment. 10 u! oltipraz produced a more pronounced effect,
but again, only under aerobic conditions (Figure 9).

35S]cysteine or [3SS]cystine by male

By one h, the uptake of [
schistosomes was significantly inhibited (41% and 36% of control,
respectively) during 12 vitro incubations in the presence of 5 1g!

35S]GSH formation occurred in the presence of 5

oltipraz (Figure 10). [
uM oltipraz, and although less label was incorporated, the relative
distribution of either labelled precursor was minimally affected.
There were no significant differences between the cysteine and cystine

controls.

[14C1glucose uptake was significantly reduced by 5 u! oltipraz
only at 60 min (Figure 11). Neither the oxy derivative of oltipraz

(100 pg) nor cysteine (1 m!) had any effect on labelled glucose uptake.

The amplitude and frequency of endogenous electrial transients in
male §;_ ‘mansoni were profoundly depressed after in yiyg exposure to
oltipraz, even at the low dose of 125 mg/kg. The onset of significant
depression occurred by 6 h, with the high amplitude (>40 uV) potentials
exhibiting a higher level of sensitivity (Figure 12). Schistosomes
exposed to 3.5 uM_ oltipraz ‘in .XiEEQ. exhibited a similar level of
depression to the drug, with high amplitude electrical activity being
significantly depressed after incubations as brief as l h. The
profbund depression of surface electrical activity observed after 24 h

incubations in 10 HM oltipraz was not observed when the parasites were

34

Figure 9: Oltipraz effect on schistosomal glutathione levels in yitgg:
Aerobic vs anaerobic environment. Paired schistosomes were incubated
as described in Figure 8 in either an aerobic or anaerobic environment,
with or without oltipraz, as indicated. Fbr anaerobic experiments, N2
first bubbled through an oxygen trap occupied the gas phase in the
flasks. values represent the means of triplicate samples of males
separated at the end of the experiment. [at] = p < 0.05 from control
value at the time period specified, [as] = p < 0.05 anaerobic value
significantly different from corresponding aerobic value. vertical

lines are l S.E.

GSH (n moles/mg protein)

 

 

29 so
Air
Control "2
18" ‘°’°M WW 4;:—
lO-sM Oltipraz 1:; * *

 

36H

*

Card 112‘-
E

10‘6M Oltipraz 1:;

3

Figure 10: [ 5S]Cysteine uptake and incorporation into schistosomes in

vitro. Male schistosomes were incubated in 2.5 ml RPMI 1640 containing

3x105

dpm [3SS]cysteine per well at 37°C, in the dark, in the pres-
ence of 5 u! oltipraz (solid symbols) or vehicle only (empty symbols).
[ c or o ] represent total uptake (disintegrations/min/male) of
[BSSlcysteine, solid line; [:3 or I ] represent the relative

percentage of total 358 as [3SS]GSH and oxidized [3SS]GSH, broken line.
10 males per well, values represent means of 4 wells per data point;
Asterisk = statistically different (p<0.05) from control uptake.
Relative distribution of label, once assimilated by the parasite, into

[3SS]GSH and oxidized [3SS]GSH was not statistically different from

control.

 

 

 

I
I
I
.I
I
I
I
I
'I
I
I
I
d

O o
5 4

30

400

20

 

(hoard

TIME

36

Figure 11: [14C1Glucose uptake into schistosomes in 111559. Male
schistosomes were incubated as‘ in Figure 10, except wells contained 3 x
105 dpn [14C]glucose per well. [0] represents untreated males, [I]
represents oltipraz-treated animals (5 11M) , [n] represents parasites
treated with the oxy analog of oltipraz (50 11M) and [0] represents
male schistosomes coincubated in 5 pp} oltipraz and 1 m_M_ cysteine.

Values represent means of 4 wells per data point; Asterisk = p<0.05.

dpm ~ molo "'

60

8

8

N
0

IO

 

 

 

 

3'0
TIME (min)

I

40

I

50

60

37

Figure 12: Surface electrical activity of male _S_._ mansoni __i_n X119:
Oltipraz effect. Mice (55 days p.i.) were dosed by gavage with 125
mg/kg oltipraz [I], 250 mg/kg oltipraz [I] or peanut oil vehicle [0]
and sacrificed at the times indicated. Values presented are the means
of 6 to 8 animals analyzed per time point per test dose; vertical

lines are l S.E. Asterisk = p<0.05.

 

 

7.2.33 . 32:3.oo

 

TIME (Hrs)

38

coincubated in 1 my cysteine or 1 mg glutathione, but not by l m}! or
100 RM 2-mercaptoethanol. The oxy analog of oltipraz (100 u!) did not

alter parasite surface electrical activity from control levels.

Tegunent potentials recorded from male schistosomes were
significantly depolarized by l u! oltipraz following 18 h in yitrg
incubation. This effect was prevented by the addition of 1 ml} cysteine
to the incubation median, whereas cysteine by itself did not alter the

tegument potential, nor did the oxy analog of oltipraz (Figure 13) .

Discuss ion:

 

Bueding st 31. [26] observed that _i_n 11332 treatment of S_._
mansoni with a 250 mg/kg dose of oltipraz resulted in a depression of
parasite GSH levels to roughly 40% of control over the first week after
treatment. In the present study, depletion of GSH was observed by l h
in both parasite and host tissues at a dosage of 250 mg/kg peg 2s. The
fact that GSH levels in parasite tissue were not rapidly restored to
control levels, as was seen in host tissues, indicates that there may
be important differences in the biochemical regulation of GSH between
the parasite and host. The present results are not in conflict with
those of Bueding _e_t_ _a_Il_ which showed depressed parasite GSH and elevated
host tissue GSH after oltipraz treatment _i_n lilo, as their experiments
did not include measurement of the acute response of host tissue or
parasite GSH levels to oltipraz. A similar depletion of parasite GSH
levels in the presence of oltipraz was observed __i_n_ Egg, allowing the

acute actions of oltipraz on the parasite to be studied independent of

39

Figure 13: Tegunent potential of male schistosomes .1-2 1122?: Effect
of Oltipraz and antagonism of effect by cysteine. Male schistosomes
were incubated in HS/RPMI containing Oltipraz or oltipraz plus cysteine
as indicated, or in the presence of 100 pl! oxy-oltipraz, for 12 h at 37
°C, in the dark. Values presented are the means of 5 animals per tegu—

mental potential average, vertical lines are 1 S.E.

 

 

-L 1 Control

* SID-GM Oltipraz

 

 

 

 

 

* -{::]10‘5M Oltipraz
-L 110-506 Oltipraz + IO'aM Cysteine
L IIO'aM Cystoino
-[ JIO-4M Oxy-Oltiproz

 

l l

-60 -55 -so -45 -4o

Togurnont Potontiol (mV)

40

the variables of host reaction to the drug.

Bueding _e_t_ _a_1_. speculated that the chemotherapeutic activity of
oltipraz may be due to competition of the drug with Y-glutamyl
cysteine, a precursor of GSH synthesis. By this mechanism the
non-stoichiometric depletion of GSH could occur at a rate dependent on
the degree of inhibition of synthesis and the turnover rate fOr
parasitic intracellular GSH. The results of the present study suggest
that there may be an alternative explanation fOr the effect of oltipraz
on GSH levels. First, the inhibition of cysteine uptake by'5 p!
oltipraz corroborates results by Seed (personal communication) that
uptake of both cysteine and glutathione, but not other amino acids, is
retarded by oltipraz. Secondly, male schistosomes were able to
incorporate [3581cysteine label into the glutathione pool, as GSH and
oxidized GSH, in the presence of 10 pg oltipraz, at a relative percent
not significantly different from control. Therefore, under the condi-
tions of acute exposure, it is unlikely that GSH synthesis is being

directly inhibited by oltipraz.

That the oxy derivative of oltipraz, which has negligible
antischistosomal effect, did not affect the uptake of [3SS]cysteine
into the parasites, even at 50 pg, suggests that the thione group of
the drug molecule is a necessary participant in the oltipraz-induced

effects on transport mechanisms.

41

In the present study, GSH (l I!!!) , cysteine (1 It!) and methionine
(1 my.) , but not 1 I!!! or 100 p! DI'I' or 2-mercaptoethanol, were able to
block the _i_n 3.1.13.9. effect of oltipraz on GSH levels occurring during
incubation. The antagonism of oltipraz effects by GSH or (SH precur-
sors may be mediated through a process of mass action on the replenish-
ment of GSH stores. DI'I‘ and 2-mercaptoethanol, unlike the other
compounds tested, are incapable of participating in the production of
GSH, which may explain their inability to antagonize the effects of
oltipraz. Alternatively, the inactivity of the oxy derivative of
oltipraz and the rather stringent structural requirements for the
maintenance of antischistosomal activity of a series of oltipraz
congeners [26] suggest that oltipraz may influence a membrane receptor,
possibly functioning as a dipeptidase or transport carrier molecule for

amino acids.

The observation that oltipraz lowered parasite GSH levels only
when the parasites were incubated in an aerobic environment indicates
that it is probably an oxidative reaction that the (SH is participating
in. Figure 14 illustrates a hypothetical scheme which could explain
the oxygen-dependency of oltipraz effect on schistosome GSH levels. If
oltipraz cycles a free radical, then organic components of the membrane
could react with oltipraz to form an organic radical. In an
oxygen-dependent reaction, an oxide radical is created which donates
the radical back to oltipraz with the abstraction of a hydrogen,
forming an organic peroxide. While schistosomes possess negligible GPx
activity, GSH detoxication of lipid peroxides occurs nonenzymatically

[100]. Similar radical cycling has been suggested for nitrofurans

42

Figure 14: Hypothetical reaction sequence to explain oxygen-dependency
of oltipraz-induced depletion of schistosomal GSH ‘32, vitro. R
represents an organic (presumably a lipid) component of the membrane,

0LT refers to oltipraz. GSH and G586 represents glutathione reduced

and oxidized , respectively.

 
  

NADH
n- I 902‘ I

transhydrogonuo

RH

   

GSSG NADPH

HYPOTHETICAL REACTION SEQUENCE RELATING
OLTIPRAZ TO GLUTATHIONE OXIDATION

43

[19], adriamycin and daunomycin [52] and paraquat [80].

Oltipraz-induced decrease in glucose uptake may be an indirect
consequence of the drug's inhibitory effect on parasite motor activity.
This is supported by the Observation that high amplitude electrical
potentials, which appear to represent summed electrical activity
originating from schistosome muscle tissue [122] , are reduced in
frequency by the drug. Thiol interference by Oltipraz could also
result in the inhibition of glucose utilization because of the wide
range of enzymes that require a thiol group as cofactor for their cata-

lytic activities [52] .

The fact that tegumental morphology is not affected by _i_n y_i_t_r_o
treatment of the parasites with 10 p_M_ Oltipraz for 12 h [23] indicates
that structural damage to the tegument is not a contributory factor in
the induction of acute oltipraz effects on membrane transport. mile
the structural integrity of the outer tegumental membrane was
maintained, microelectrode recordings showing depolarization of the
tegument suggest that a significant redistribution of ions across the
tegumental membrane had occurred. The effect(s) of a drug-induced
ionic imbalance on the Na—driven carrier mediated cotransport of
glucose [131] may have contributed to the decrease in glucose uptake
observed. In a similar manner, perturbations of ionic equilibria
across the tegument may contribute to the inhibition by oltipraz of the
uptake of GSH precursors that was observed. Consistent with the
effects on other parameters measured, tegument potentials of worms

incubated in 1 11!} cysteine or 10 p! oltipraz plus 1 my. cysteine were

44

not significantly different from control values. These results
indicate that cysteine is able to prevent Oltipraz-induced ionic imbal-
ances .ig"yit£g_ without directly affecting ionic fluxes across the
membrane. The fact that the oxy derivative of oltipraz did not affect
the tegumental membrane potential further suggests that a disruption of
membrane conductance may contribute to the antischistosomal efficacy of

Oltipraz.

Oltipraz is a very lipophilic compound which is rapidly
accumulated by the membranes Of the parasite, suggesting that there is
a hydrophobic nature to the site of drug action. Intercalation Of drug
into the tegument and muscle membranes may' partially explain the
effects Of Oltipraz on tegument potential and surface electrical
activity, in a manner reflecting the correlation between the solubility
of drugs in membranes and their ability to fluidize and disorder

‘membrane structure [110,121].

From a therapeutic perspective, the report by Ali .33 .gl. [3]
which showed that coadministration of cysteine and Oltipraz to monkeys
resulted in an enhanced bioavailability of the drug, suggests that the
‘ig .yiyg_ synergistic action of cysteine on oltipraz schistosomicidal
effects may be due to this enhancement of oltipraz bioavailability. As
the synergistic interaction of cysteine with Oltipraz may be indirect
and removed from the site of drug action, the .in .yiE£g_ evidence
presented which show cysteine antagonistic to Oltipraz action does not
refute the $2 2129 Observations. Instead, these findings suggest that

although cysteine, GSH or methionine ameliorate oltipraz action acutely

45

when applied directly to the parasite, in the long-term _i_n vivo
situation, the enhancement of oltipraz bioavailability is more relevant

clinically in the treatment of the disease.

cm THREE: RELATIONSHIP OF SCHISTUSOME GLUTATHIONE LEVELS .10

VARIOUS PHYSIOLOGIC PARAMETERS OF THE PARASITE

 

Introduction;

Schistosome GSH levels were manipulated by various compounds in
order to determine dose-dependency of acute effect and to determine if
these effects were also antagonized by cysteine, methionine and/or
glutathione in like fashion to the antagonism of oltipraz-induced GSH

depression in the parasites.

Results: BCNU, an alkylating agent which inactivates GRed [49],
effected a dose-dependent depletion Of parasite GSH levels after 3 h
incubation (Figure 15). Microelectrode recordings of schistosome tegu-
mental potential revealed that ionic conductance is also altered by
BCNU in a dose-dependent manner, an effect which is partially reversed
by coincubation of the parasites in l m! cysteine (Figure 16).
Tegumental potentials fOr 1 mM_ cysteine—treated control, fOr l m!
cysteine plus 3 m! BCNU-treated parasites, and fOr 3 mg BCNU-treated

animals were statistically different from each Of the other groups (P

(0.91).

46

47

Figure 15: Dose-dependent depletion of schistosomal glutathione by
BCNU. Wenty adult male schistosomes were incubated in 2.5 ml RPMI
containing various concentrations of BCNU, as indicated. Incubations
were performed at 37 °C, in a dark chamber, for 3 h. Values represent
two or three separate experiments with triplicate groups of 20 animals

per group. Vertical lines are i l S.E., Asterisk = p < 0.05.

CONTROL

 

 

 

 

 

A.-2_m.pozo oz.

330sz mzoib‘gu

no
no
0

3!)

L0

133

(MM)

BCNU

48

Figure 16: Tegument potential of male schistosomes _i_n_ 13319: Effect
of BCNU and antagonism of effect by cysteine. Male schistosomes were
incubated in HS/RPMI containing BCNU or BCNU plus cysteine as
indicated, for 12 h at 37°C, in the dark. Values presented are the
means of 5 animals per tegumental potential average, vertical lines are

l S.E.

 

-L 1 Control
: lmM Cysteine + 3mM BCNU

-l 3mM BCNU
£22.3me BCNU

L l L j l

-50 '40 -3O -2O " IO

Tegurnent Potential (HIV)

49

BSO, an inhibitor of Y-glutamyl cysteine synthetase [60], produced
a dose-dependent depletion of GSH in male §;_ mansoni. The decline in
GSH levels (Figure 17) was less dramatic than results obtained with
diamide or BCNU and required a longer incubation period. NO acute
differences from control were observed in longitudinal muscle tension
development over the first 30 min of exposure to 1 m}! B80. B80 at 100
pH for 18 h produced a significant depression of high ampliture (> 40
pV) electrical potentials recorded from the surface Of male parasites
(93 :_ 47 potentials ESQ—treated, 253 :_ 81 potentials control).
Coincubation in the presence of l mM_cysteine inhibited BSD—induced
depression of electrical activity (262 :.92 potentials). Surprisingly,
Bsobtreatment at 1 mg resulted in egg counts not different from
controls over the 72 h period, although GSH levels fOr the paired worms
were depressed 30-40% from control levels at 72 h. Oltipraz effects on
schistosome fecundity in yitgg are shown in Figure 18. The antagonism
of Oltipraz effects by various compounds in the same assay is shown in

Figure 19.

Discussion:

 

BCNU irreversibly inactivates GRed. This inhibition is selective
as BCNU does not inhibit either the enzymes of glucose catabolism or
GPx activity [49]. The prevention by cysteine of GRed inactivation by
BCNU [49] may explain the antagonism of BCNU-induced depression of
schistosomal tegumental potentials observed in this study,
alternatively, as cysteine feeds into GSH synthesis, these data are

also supportive Of the hypothetical reaction sequence shown in. Figure

50

Figure 17: Dose-dependent depletion of schistosomal glutathione by
BSO. Twenty adult male schistosomes were incubated in 2.5 ml RPMI con-
taining various concentrations of B80, as indicated. Incubations were
performed at 37 °C, in a dark chamber, for 6 h. Values represent two or
three separate experiments with triplicate groups of 20 animals per

group. Vertical lines are i l S.E., Asterisk = p < 0.05.

CONTROL

 

*

I

I
3.0

[.0

 

 

 

I” o.
O O

0.3

2.
O

A7339: 92.330sz $.25...st

BSO (MM)

51

Figure 18: Oltipraz inhibition Of schistosome fecundity .ig_'yitgg.
Paired schistosomes (15 pair per 50 ml HS/RPMI in 250 ml Erlenmeyer
flasks) were incubated 72 h at 37‘C in the presence or absence of
Oltipraz at the concentrations indicated. Asterisk = p < 0.05 from
control. values presented are the means of 5 to 7 flasks run at each

concentration; vertical lines are l S.E.

No. of eggs per female in 72 hours

 

 

 

 

o 59 I90 I§O 290
Control I'-
Io‘7 M OI:
4xIO'7 M on as

Io‘6M OI: *

Figure 19: Effect of various reagents on oltipraz inhibition Of
schistosome fecundity iguyitgo. Paired schistosomes were incubated as
described in Figure 18. +OLT indicates coincubation of parasites in
0.4 pg oltipraz, McEtOH is 2— mercaptoethanol, DTT is dithiothreitol
(Cleland's reagent), CYST is cysteine, METH is methionine and GSH is
glutathione, reduced form. [*1 = p < 0.05 from control, [at] = P <
0.05 from Oltipraz only, i.e. statistically significant prevention of
oltipraz effect. values presented are the means of 5 to 7 flasks run

at each concentration; vertical lines are l S.E.

No. of eggs per female in 72 hours
L 5.0 19° "5.0

 

 

Control
OI t *

II

10% Menomon *

101M DTT *
D" + O" *

 

IO‘3M METH+Olt

lO‘3M osn
asu.on_——E‘”

 

 

14.

Despite the toxic effects of BCNU on the schistosome, there is
little chemotherapeutic potential for this particular compound. BCNU
is an anticancer agent which exhibits mammalian toxicity due to alkyla—
tion of biological macromolecules. Thus, while BCNU may selectively
jeopardize the schistosome because of its absence of GPx activity, the
carcinogenic potential of this drug would lhmit its usefulness. As
BCNU appears to be able to inactivate GRed without metabolic activa—
tion, its mechanism of action may not be related to alkylation, hence
there may be potential fOr a BCNU-like compound if the enzymic inhibi-
tion properties can be dissociated from the alkylation potential

inherent in BCNU.

BSO requires intracellular activation to BSO—phosphate before
inhibition of Y—glutamylcysteine synthetase occurs. If the plateauing
of GSH depletion at 6 h due to BSO concentrations greater that 1 mg
indicates near total inhibition of the enzyme, the slow rate of GSH
decline suggests that the turnover rate 12 21259 is on the order Of
12.5 h. TUrnover rates in the literature vary from estimations Of 0.5
h fOr rat kidney to 65-96 h for mammalian erythrocytes. Tissue culture
and tumor cells exhibit biological half—life fOr GSH in the range of

6-8 h [87].

54

Methionine, cysteine and GSH were observed to provide significant
ameliorative action on egg laying inhibition due to Oltipraz. Both
methionine and cystein e may be used fOr the intracellular enzymatic
synthesis of GSH, which may aid in the maintenance of GSH levels,
whereas DTT and 2—mercaptoethanol do not contribute to the synthesis of
GSH. The poor correlation between GSH levels and fecundity was
unexpected, however it may be an artifact of the technique employed, as
worms from several flasks were pooled in order to assay for GSH.
Alternatively, lg ZiEEQ culture dramatically affects oviposition in
effecting a lower output of eggs, especially during the first 24 h
period, when the worm pairs are "in shock" while they adjust to the new
media and nonphysiologic conditions [unpublished observations]. As
many factors may be affecting the reproductive system which do not
influence GSH levels, the amelioration of oltipraz effects on schisto-

some reproduction may be merely a qualitative finding.

CHAPTER FOUR: EFFECTS OF SCHISTOSOMAL INFECTION ON HOST REGULATION OF

GLUTATHIONE LEVELS

Introduction:

 

The cause of the major disease syndromes of schistosomiasis is the
retention of a large number of eggs in host tissue [134]. Most of the
eggs freed into the circulation are sieved out by the liver where host
granulomatous inflammatory response to the eggs results in a
presinusoidal blockage of portal blood flow, as well as hepatic

necrosis and subsequent fibrosis.

Monoamine oxidase activity, a sensitive indicator of liver
fibrosis [72], is significantly depressed in mice by the 6th week
following infection with §;. mansoni [1]. There is also a marked
depression of microsomal drug-metabolizing enzyme activities of the
host liver. A close correlation exists between the onset and degree Of
enzymic depression with the severity and age of infection [32,33]. In
the metabolism of xenobiotics, the tripeptide glutathione (GSH) plays
an important role in chemical detoxication processes [52].
Experhmental GSH depletion exacerbates toxicity due to protein alkyla-
tion or lipid peroxidation caused by chemically reactive, electrophilic
chemicals or metabolites [4,77]. Glutathione peroxidase (GPx) is an
integral part of the cellular antioxidative system [36], reducing
peroxides in a reaction with GSH, which becomes oxidized in the
process. Glutathione reductase (GRed) reverts oxidized GSH to its

reduced state at the expense of NADPH. The glutathione S—transferase

55

56

(GTr) family of enzymes removes potentially cytotoxic alkylating agents
through conjugation with GSH [21,84]. GTr also have GPx activity [113]
which suggests a secondary role in detoxication of the byaproducts of

oxygen utilization.

As the toxicity Of antiparasitic drugs to the host may limit the
therapeutic value of these compounds, changes in the ability of the
host to detoxify xenobiotics due to the presence of parasitic infec-

tions are of considerable importance.

Results:

GSH levels in host liver decline during the course of the infec-
tion, while kidney cortex levels remain constant (Figure 20). Hepatic

GSH is statistically depressed from uninfected liver values by day 50.

Hepatic enzyme activities are summarized in Table 3. The GRed
activity of infected mouse liver was not significantly different from
uninfected controls at 50-60 days, however GPx enzymic activity was
depressed to 37% Of uninfected levels. Hepatic GTr from infected mice
exhibited 26% of uninfected liver activity. Neither renal GPx nor GRed
enzymic activities were altered due to schistosomal infection. Kidney

cortex was not examined fOr GTr activity.

57

Figure 20: Glutathione levels in host hepatic and renal cortical
tissues during the course of Schistosoma mansoni infection. Means of
triplicate samples of organs where the host harbored 15-30 pairs of
parasites, n=5 animals per date, [I] represents hepatic GSH, [I]
represents renal cortical GSH. Asterisk = statistically different
(p<0.05) from uninfected control values at the time period specified,

vertical lines are l S.E.

nmoles GSH - mg protein ‘4

 

 

 

150 «
1.00 ~
0.50 «
30 40 so 60 7o 80 90

AGE (Days Post—Infection)

58

Table 3: Mouse hepatic glutathione—related enzyme activity:
Effect of Schistosoma mansoni infection.

Hepatic enzyme activity

 

 

GPx 1 GRed 2 GTr 3
Uninfected liver 440143 6617 199126
Infected liver 161139 * 62111 ns 52122 *

1 - Glutathione peroxidase activity, with t—butyl hydroperoxide as

substrate, is expressed as nmoles NADPH oxidized/min/mg protein.
2 - A unit of glutathione reductase activity represents the oxidation
of 1 pmole NADPH/min/mg protein, results are expressed as mU.

3 - Glutathione S—transferase activity is expressed as nmoles product
per min/mg protein, after correction for spontaneous nonenzymatic
conjugation, p—nitrobenzyl chloride was the substrate.

* - Statistically different (p<0.05) from uninfected liver value, ns=
not significant as determined by Student's t—test.

59

Discussion:

 

The relative contribution of GSH depletion or the depression of
hepatic enzyme activity to the hepatopathology of schistosomiasis could
depend, in part, on the redundancy of the glutathione system. A func-
tional reserve capacity fOr GPx [127] and GRed [49] has been shown in
mice. Further, as the GTr are a family of enzymes with overlapping
substrate specificities, and constitute 5-10% of the liver cytosol
protein [25], a functional reserve capacity for mOuse liver GTr is also
likely. In addition, y—glutamylcysteine synthetase activity' may
increase as GSH levels decline [76]. However, the rate of synthesis of
GSH in the infected animals was inadequate to maintain GSH at control
levels, despite possible redundancy in the system. These results
suggest that animals already compromised by a schistosomal infection
may have enhanced susceptibility for hepatic toxicity from xenobiotic
compounds which impose either an oxidative stress or require conjuga-

tion fOr detoxication.

SUMMARY AND CONCLUSIONS

The present study indicates that the prolonged antischistosomal
activity of oltipraz may be due, in part, to the acute effects observed

.12 vivo and .15 vitro. Disruption of tegumental function in the

 

transport of glutathione or glutathione precursors into the schistosome
effects a depletion of intracellular GSH stores. As was observed fOr
other sulfhydryl inhibitors [73], the ionic gradient across the tegu-
mental membrane is disrupted by oltipraz. These effects of the drug on
the tegument and the subsequent depletion Of schistosomal GSH lead to
the depression of basic physiological parameters (frequency and
amplitude of surface electrical potentials and inhibition of
reproductive processes) and the alteration of biochemical processes,
including indirect effects on glucose utilization. The present study
also indicates that GSH and GSH precursors inhibit the acute actions of
oltipraz '12 ‘yiggg. A likely hypothesis fOr the synergistic action of
cysteine on the schistosomicidal activity of Oltipraz .12 .2129 is
related to cysteine's enhancement of the bioavailability of the drug
[3]. That DTT and 2-mercaptoethanol do not antagonize GSH effects .13
.yipgg is probably related, in part, to the fact that these compounds

cannot serve as precursors to d9 novo GSH synthesis.

60

61

Concerning the enzymes of glutathione regulation, one of the major
findings of this study is the elucidation of biochemical differences
between the host and the parasite in the level of enzymic activity
present in GPx, GRed, our and Y-GTP. These differences may be exploit-
able chemotherapeutically. The fact that the parasite has different
enzymic activities in regard to GSH utilization is not surprising
because of the difference in the parasites metabolic handling Of
xenobiotics compared to the host. Further, the parasites do not
require oxidative reactions to provide energy, and therefOre, do not
apparently require similar levels of GSH in order to protect against

oxidant injury.

In light of the hepato—pathology of the disease, changes in the
biochemical regulation of GSH stores were not unexpected. These
changes may indicate that the host is more at risk as the liver is the

major organ for detoxication of most xenobiotics.

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