133

Mutatmn Research, 58 (1978) 133--142

Elsevier/North-Holland Bloinedical Press

ABSENCE O F MUTAGENICITY OF P R A Z I Q U A N T E L , A NEW,

EFFECTIVE, ANTI-SCHISTOSOMAL DRUG, IN BACTERIA, YEASTS,
INSECTS AND MAMMALIAN CELLS

H. BARTSCH 1, T K U R O K I 1, C M A L A V E I L L E 1, N LOPRIENO 2, R. B A R A L E 2,
A. A B B O N D A N D O L O 3, S. B O N A T T I 3, G R A I N A L D I 3, E. VOGEL 4 and A. DAVIS s

1 Unit o f Chemical Carcmogenesls, Internatmnal Agency for Research on Cancer, Lyon

(France), 2 Laboratorzo dl Genet~ca, Unwers~ty o f Pzsa, Ptsa (Italy), 3 Laboratorm d~
Mutagenesz e Dzfferenz~amento, C N R , P~sa (Italy), 4 Department o f Rad~atmn Genetics
and Chemical Mutagenests, State Unwerslty o f Le~den, Lezden (The Netherlands), and
s Umt o f Schlstosomtas~s and other Helmmth~c Infectmns, Dwzsmn o f Malarza and other
Paras~tzc Dzseases, WHO Headquarters, Geneva (Switzerland)

(Received 5 April 1978)

(Revision received 26 May 1978)
(Accepted 5 June 1978)

Summary

Prazlquantel (Embay 8440, Droncit) a new, effectwe anti-schistosomal drug,

was tested in various short-term assays that have shown a predictive value for
the detection of potential carcinogens. Indicator organisms S. typhimurmm
stratus, S. pombe, S. cerews~ae, cultured V79 Chinese hamster cells or human
heteroplold cells and Drosophila melanogaster were treated with Prazlquantel.
The reduction of reverse and forward mutahons, mitotic gene conversions,
X-hnked recessive lethals, slster-chromatld exchanges and unscheduled DNA-
repmr synthesis was scored; rodent-liver microsome-, cell- and host-mediated
assays were also performed. Hycanthone, another schlstosomicide was included
as a positive control. The absence of a genetic activity of Praziquantel uni-
formly observed m such a battery of tests (1) confirms the assumption that the
anti-schlstosomal effectiveness of this drug is not related to the mutagenic
activity and (11) should encourage the implementation of extended clinmal and
field trials.

Abbrevlatzons DMSO, chmethylsulphoxtde, MC, 3-methylcholanthrene, PB, phenobarbltone, S-X,

X X 1000 X g hver supernatant
134

Introduction

Helmmthlc refections, whmh are spreading in m a n y countries, are among the

major causes of morbidity and physmal disablhty m man in the developmg
countries of the tropms; they are also of great economm importance when they
occur in domestm animals [1--4]. An increase m schistosomiasis m tropmal
endemm areas, in partmular, seems to result from those economic development
schemes that involve man-made lakes or other irrigation systems [5,6].
Although crude estimates suggest that 200 milhon persons are affected by
schistosomiasis [7], none of the currently available schistosomicldes meets all
desirable criteria for use in mass-chemotherapy programmes, i.e. high curative
efficiency against the different stages of the 3 c o m m o n schlstosomes infecting
man, high patient-acceptance rate, mmlmal treatment time, absence of adverse
general blologmal effects and acceptable cost. Some antl-schistosomal drugs,
such as nindazole and hycanthone, are mutagenic and carcinogenm [8,13].
Furapromidium, widely used in China [14], is also mutagenic [15]. There is
evidence, however, than anti-schistosomal and mutagenic properties of a drug
may n o t be associated [16,17].
Praziquantel (EMBAY 8440, Droncit R, 2-cyclohexylcarbonyl-[1,3,4,6,7,
llb]-hexahydro-2H-pyrazine[2,1-a]isoquinoline-4-one) is a newly developed
drug (Fig. 1) which is not structurally related to the above-mentmned com-
pounds or the other schistosommldes m use [18]. It is highly active against a
wide range of cestodes and against all species of schistosomes that are patho-
gemc to man [19,20]. In initial clinical tnals, single-dose or single-day treat-
ments of human infectmns due to Schistosoma haematobium showed a high
therapeutic effect: of 80 treated Zambian children, 66 were followed to one
year and only 2 parasitological failures were detected and no adverse effects on
the haemopomtic systems were observed (ref. 22 and A. Davis, unpublished
results). On oral dosage, Praziquantel was rapidly metabolized and excreted
predominantly in the u n n e [23].
Following advice from WHO, Geneva, we initiated a collaborative mvestlga-
t m n to test Praziquantel for its ability to reduce genetic effects in S. typh~mu-
hum, Schizosaccharomyces pombe, Saccharomyces cerewszae, Drosophila mela-
nogaster and cultured V79 Chinese hamster cells in the presence or absence of a
metabohc activation system. These short-term tests have shown a predictive
value for the detection of potential carcinogens [24]. We report here a sum-
mary of the results in which, for the assays zn v~tro, only values for the highest

Fig 1 Chemical formula of Prazlquantel

 135

concentration of Praziquantel tested are listed, since similar results were

obtained with various lower concentrations. The results lndmate the absence of
any notable mutagemc effect of Praziquantel, whereas hycanthone, included as
a positive control in some of the assays, was biologically active.

Materials and methods

Prazlquantel (purity >99%), was a product of Merck, Darmstadt, and Bayer

A.G., Leverkusen, Federal Republic of Germany. The experiments (1--25)
listed in Table 1 were carried out as described below.
Expts. 1,2. The presence of the R factor in S. typh~murium strains TA100
and TA98 and tl~elr mutability were checked [25]. 9000 g liver supernatants
(S-9, 25% w/v in 0.15 mM KC1--5 mM SOrensen buffer, pH 7.4) were ob-
tained from female BD-VI rats or from male OF-1 mice. Groups of 2--4 ani-
mals received 1 1.p. injection of Aroclor 1254 (500 mg/kg bw) 5 days before
they were killed or of 3-methylcholanthrene (40 mg/kg) 2 days before, or
received a combined treatment of MC and phenobarbitone (0.1% in drinking
water for I week). S-9, cofactors (NADP , G-6-P, Mg2), bacteria (1--2 10 s
cells/plate) and a solution of Praziquantel in DMSO (100 pl) were combined in
hlstidine-poor soft agar (0.55% w/v Difco agar, 0.55% w/v NaC1, 45.5 pM his-
tidme and 45.5 pM biotin in 5 mM SCrensen buffer, pH 7.4) and plated in trip-
licate. The number of h~s revertant colonies after 48-h incubation at 37 was
scored [26]. In some assays with the TA98 strain, 1,1,1-trichloropropene-2,3-
oxide was added to a final concentration of 0.4 mM [27]. Mean values -+ S.E.
of 3--5 experiments are listed.
Expt. 3. Yeast (P1 strmn SP-98 ade 6--60/rad 10--198/h- haploid strain of
S. pombe) was incubated in the presence of Praziquantel dissolved in butan-2-ol
(final concentration, 0.5% v/v) from male Swiss albino mice liver S-9 and cofac-
tors (NADP , G-6-P, Mg2+). Forward mutations at 5 adenine loci were scored
[28].
Expts. 4--7. Diploid yeast strain D4 of S. cerevis~ae was incubated in the pres-
ence or absence of a mouse liver S-9 and the test c o m p o u n d dissolved in butan-
2-ol. Mitotic gene conversions at the trp5 and ade2 loci were scored [28]. Mean
values + S.E. of Independent experiments are listed.
Expts. 8--10. In the host-mediated assay, 5 107 cells of either S. pombe or
S. cerewsiae were inoculated intravenously into male Swiss albino mice (25 g
bw). Groups of 3 animals were treated by gavage with Praziquantel at 300
mg/kg bw twice with a 3-h interval. Control animals were inoculated with yeast
cells only. After 5--24 h, yeast cells were isolated and scored for forward muta-
tions or gene conversions [28].
Expts. 11,12. The unscheduled incorporation of 3H-TdR and liquid-scintilla-
tion counting were used to estimate DNA-repair synthesis m cultured human
heteroploid cells (EUE) [29]. The cells (2.5 l 0 s) were seeded on 50-mm dishes
containing 6 coverslips (12 mm 12 mm), grown overnight and then treated
m a monolayer for 1 h with Praziquantel dissolved in DMSO (final concentra-
tion 0.5% v/v) in 3 ml of either a reaction mixture containing 10 000 g liver
supernatant (S-10) from male Swiss albino mice and cofactors [30] or Hanks's
balanced salt solution. Cells were then exposed to 3H-TdR (10 #Ci/ml for 4 h in
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138

the presence or absence of 5 mM hydroxyurea. Tritium incorporation into the

acid-insoluble fraction was counted in a toluene-based scintlllatson cocktail in a
Packard trl-carb spectrometer. Results are expressed as the percentage ratio of
incorporated 3H-TdR in the presence and absence of hydroxyurea.
Expts. 13,14. V79 Chinese hamster cells (5 105) were seeded in Falcon
flasks (25 cm 2) and grown overnight. Prazlquantel dissolved m DMSO (final
concentration 0.5% v/v) was added to 5 ml of either a reaction mixture con-
talnmg S-10 fraction from liver from Swiss albino mine and cofactors or Dul-
becco's modified MEM. After 3 h incubation at 37C, slster-chromatld
exchanges were scored after 2 cell cycles in the presence of BUdR (3 pg/ml)
[31]. Mean values -+ S.E. from 20 metaphases are hsted.
Expts 15,16 V79 Chinese hamster cells (overnight cultures after plating
1.5 106 cells per 25-cm 2 flask) were incubated for 1 h at 37C m 5 ml of
either a reaction mixture containing S-10 liver fraction from male Swiss albino
mice and cofactors or Hanks's balanced salt solution, to whmh Praziquantel dis-
solved in DMSO (final concentration 0.5% v/v) was added. After 4 expression
times (0, 90, 114, 138 h), colonies resistant to 6-thloguanme (5 pg/ml) were
scored after 14 days [30].
Expts 17--20. V79 Chmese hamster cells (overmght culture after platmg
106 cells per 60-mm dish) were incubated for 3 h in 2.5 ml of a reaction mLxture
containing 30% w/v of a 15 000 g liver supernatant (S-15) from phenobarbi-
tone-pretreated BD-VI rats (0.1% m drmkmg water for 7 days) in the presence
or absence of cofactors (NADP ~, G-6-P, Mg~) and Prazlquantel dissolved m
DMSO (final concentration, 0.8% v/v), followed by post-incubation m fresh
culture m e d m m for 2--3 h and plating for determination of mduced cytotoxlclty
and 8-azaguanine- (20 pg/ml) and ouabmn- (1 pmol/ml) resistant mutants. The
drugs were added 48 h after plating [32].
Expts. 21-24. In cell-medmted assays [33], rat-embryo cells at secondary
culture were irradiated with 4000 R and plated at 2.5 104 cells per 60-mm
dish V79 Chinese hamster cells were plated on top of the feeder layer at 3
10S/dish, and 5--6 h later Prazlquantel dissolved m DMSO (final concentratlon~
0.5% v/v) was added for 48 h, followed by platmg for determinatmn of induced
cytotoxiclty and of 8-azaguanme- and ouabain-resistant m u t a n t colonies as m
Expts. 17--20.
Expt. 25. X-hnked recessive lethal mutations were measured in Drosophila
melanogaster, Berlin K strain, by feeding 2-day-old males on glass filters [34],
with an aqueous solutmn of 2% DMSO, 5% sucrose and various concentratmns
of Praziquantel. After treatment, each male was mated with 3 or 4 virgin Basc
females [35] for 4 consecutive broods at 2- or 3-day intervals for a total period
of 10 days. F2 cultures with more than one wild-type male were considered to
be non-lethals. Further tests were carrmd out to confirm that a suspected cul-
ture carried a recessive lethal m u t a t m n . The percentage lethal mutations + S.E.
were calculated from the number of lethals per gamete tested, 2 experiments
being used for each concentration.
Results and discussion
In the plate-incorporation assay [26], m the presence of S typhzmurzum
stratus TA100 and TA98 (Expts. 1, 2), concentratmns of up to 2 mM Prazl-
 139

quantel did n o t increase the number of reverse mutations to his*, either in the
presence or absence of an S-9 hver fraction from rats or mice pretreated with
either MC, Aroclor or combined MC and PB. As Praziquantel has an aromatic
ring which could be oxidized by microsomal mono-oxygenases to yield reactive
arene oxides, we tried to inhibit their hydration to dihydrodiols. 1,1,1-Tn-
chloropropene-2,3-oxlde is such an inhibitor of mmrosomal epoxide hydratase
and potentiates the mlcrosome-mediated mutagenlcity of polycychc hydrocar-
bons [27]. Its addition at a concentration of 0.4 mM to plates contmnmg the
TA98 strain and S-9 liver fractions from rats or mice pretreated with MC or
Aroclor did n o t increase the number of his+ revertant colonies.
Praziquantel, at a concentration of 5 mM did n o t increase the number of
forward mutations in S. p o m b e (Pl-straln) at 5 adenine loci (Expt. 3) after 4-h
incubation m the presence of an S-9 mouse-hver fraction. Similarly, no mitotic
gene conversions at the ade 2 or trp 5 loci were induced in S. cerevzsiae, D4
strain (Expts. 4--7), in the presence of an S-9 mouse-liver fraction for 4 h or in
the absence of an activation system for 24 h incubation with a concentration of
10 mM Praziquantel, although a concentration of 0.5 mM h y c a n t h o n e at pH
6.5 increased the conversion frequency to 10 times the spontaneous level at the
two genetic loci d u n n g a 6-h Incubation period.
In the host-mediated assay in mice (Expts. 8--10), Prazlquantel at 600 mg/kg
bw did n o t produce a statistically signlfmant increase in the frequency of for-
ward mutations in S p o m b e or of gene conversions in S. cerevisiae following
1.v. Injection of the yeast cells (results from 2 experiments are listed).
Unscheduled incorporation of 3H-TdR, in the presence of h y d r o x y u r e a to
suppress DNA replication, was used to estimate DNA repair synthesis in a cul-
tured human heteroploid cell line (EUE) (Expts. 11,12). Dunng 1 h of incuba-
tion, up to a 5 mM concentration, Praziquantel, in the presence or absence of
an S-10 mouse-hver fraction, induced no DNA lesions t h a t led to DNA-repmr
synthesis; whereas 1 mM hycanthone, in the absence of an S-10 liver fraction,
increased the incorporation of 3H-TdR 3 times over the control level.
No slster-chromatid exchanges (Expts. 13, 14) were induced in cultured V79
Chinese hamster cells in the presence or absence of an S-10 mouse-hver frac-
tion, at concentrations up to 1 mM PrazIquantel during 2 h of incubation.
Under these conditions, 0.1 mM hycanthone increased the number of sister-
chromatid exchanges per metaphase to 7 times the spontaneous level.
A concentration of up to 3 mM Praziquantel did n o t induce 6-thloguanme-
resistant mutants in V79 Chinese hamster cells (Expts. 15, 16) during 1 h of
treatment e~ther in the presence or absence of an S-10 mouse-hver fraction,
1 mM h y c a n t h o n e increased the mutation frequency 60-fold over the sponta-
neous level.
The mutageniclty of Prazlquantel was also examined in V79 Chinese hamster
cells in the presence of an S-15 liver fractmn from phenobarbitone-pretreated
rats (Expts. 17--20) and in cell-mediated assays (Expts. 21--24), by the use of
mutations to 8-azaguanine and ouabam resistance. In the microsome-mediated
assay, exposure to up to 1 mM PrazIquantel for 3 h induced no toxmlty and no
azaguanme- or ouabmn-reslstant m u t a n t colonies. Polycychc hydrocarbons are
more effmiently detected as mutagens in cell-mediated assays whereby V79
Chinese hamster cells are co-cultwated with lethally irradiated rodent emb~,o
140

cells as a feeder layer [33] ; in this assay, Prazlquantel at concentrations greater

than 0.5 mM caused growth inhibitmn during the 48-h incubatmn period, but
no 8-azaguanme- or ouabmn-resistant m u t a n t colonies were observed in the
presence or absence of a feeder layer.
Praziquantel was also tested for reduction of X-hnked recessive lethal muta-
tions in Drosophila melanogaster, m two large series of experiments m which
Prazlquantel was given at 2 or 3 concentrations in each series (Expt. 25). The
results gave no indication of a mutagenic effect of Prazlquantel in Drosophila,
when the data from all treated groups were pooled, the percentage of recessive
lethal mutations obtained was 0.25 -+ 0.05 (30 lethals m 11 788 gametes tested),
whmh was the same as the spontaneous m u t a t i o n frequency.
Prazlquantel thus had no detectable genetm actlwty in varmus short-term
tests, with different genetic indicators, different bmlogmal end-points and
metabohc actlvatmn systems in vlvo and m vitro. Our results confirm the
reported absence of mutagenlclty of Prazlquantel m Salmonella typhtmunum
in tissue, host- and unne-medlated assays [36] and also in dominant-lethal and
micronucleus tests in mice and in the spermatogonlal tests in Chinese hamsters
[37]. The absence of mutagemc activity of Prazlquantel, whmh was uniformly
observed, further confirms the assumptmn that an antl-schlstosomal effec-
tiveness is not necessarily related to the mutagenm achvlty of a drug. However,
the negative results of this battery of tests cannot y e t be taken as proof that
Prazlquantel has no carcmogemc achvity and long-term carclnogenmity assays
In rodents are at present under way (U. Mohr, personal communication)

Acknowledgements

Prazlquantel was a gift of Merck, Darmstadt, and Bayer A.G., Leverkusen,

Federal Republic of Germany. S typh~murtum strams were generously pro-
vlded by Professor B.N. Ames, Berkeley, Cahfornla, U.S.A. Chmese hamster
cells for experiments 12--24 (Table 1) were obtained from Dr. E. Huberman,
Oak Ridge National Laboratory, Oak Ridge, Tennessee, U.S.A. The authors
would hke to acknowledge the skilled technical assistance of Mrs. G. Brun and
to thank Miss L. Kitchen for secretarial assistance, Mrs. E. Heseltlne for
editorial aid and Drs. L. Tomatls and R Montesano for cntmal reading of this
manuscript. Fmancml support for the authors' research activities was partially
provided by WHO, Umt of Schlstosommsls and other Helmlnthlc Infections,
Division of Malaria and other Parasltm Diseases, and by the Commlssmn of the
European Communitms.

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