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Effect of hycanthone on Schistosoma mansoni macromolecular synthesis in vitro
Molecular and Biochemical Parasitology, 2 (1981 ) 295 - 307
295
© Elsevier/North-Holland Biomedical Press
EFFECT OF HYCANTHONE ON SCHISTOSOMA MANSONI MACROMOLECULAR SYNTHESIS IN VITRO
L ~ ' I A PICA ]dATTOCCIA, ALESSANDRA L E L I ' and DONATO CIOLI
Laboratory of Cell Biology, National Research Council, 18 Via Romagnosi, 00196 Rome, Italy (Received 14 April 1980; accepted 14 August 1980)
Adult, immature and hycanthone-resistant schistosomes were allowed to incorporate tritiated precursors of maeromolecule ~ynthesis in vitro, either in the presence of various concentrations of hycanthone, o~" at various times after removal of the drug. The effect on worms was compared to that on Hel.a cells. The results show that hyeanthone markedly inhibited the incorporation of uridine in all the sy ,terns studied, while the incorporation of thymidine and leucine was only secondarily affected. TI~c inhibition of uridine incorporation reflected in part a decreased uptake of the radioactive precursor. ' f ' z hycanthone-induced inhibition of uridine incorporation was essentially irreversible upon remo :~t[ of the dxug ill adult sehistosomes, while it was completely reversible in hycanthoneresistant wol.as, in immature worms and in HeLa cells. The effects of a hycanthone analog, IA-4, were largely comparable to the effects of the parent compound. These results suggest that the inhibition of RNA synthesis can be a possible explanation for the mechanism of the schistosomieidal action of hycantho~e. Key words: Hycanthone, Hycanthone analog IA-4, RNA synthesis, Drug mechanism, In vitro labeling,
Schistosoma mansoni.
INTRODUCTION
Hycanthone is one of the most widely vsed drugs in human infections with Schistosoma ma,2soni and S. hematobium. It is one of the metabolites of a previously used compound, lucanthone or Miracil D [ 1 ]. The mechanism of its schistosomicidal activity is not dear, although hypotheses have been advanced on its possible interference with the functioning of neurotransmitters in the worms [2, 3 ]. The hvcanthone molecule contains a planar aromatic triple ring system which can interact w~th DNA [4] probably by intercalating between adjacent base pairs [5]. Presumably connected with these biochemical properties, hycanthone is mutagenic [6], teratogenlic [7] and possibly carcinogenic [8, 9]. The effects of hycanthone and lucan-
~bbreviatic,ns: TCA, trich!oroacetic acid; IA-4, 8-ch!oro-2[2-(diethylarnino) ethyll-2H-[ 1 ]benzo~iopyrano.[4, 3, 2-cd ]..indazole-5-methanesulfonate.
296
thone on nucleic acid and protein synthesis have been studied in a number of bacterial and mammalian systems [10-13]. In general, a pronounced but reversible inhibition of RNA synthesis was observed, while DNA and protein synthesis were affected to a lesser and more variable extent. On the basis of the above-mentioned data, the possibility should be considered that the schistosomicidal action of hycanthone might be due to its interference with the macromoleeular synthesis of the parasite. Surprisingly, however, no direct studies were available, until recently, on hycanthone effects on schistosome nucleic acid and protein synthesis. A recent communication [ 14] actually reports an inhibition of [3H]adenosine incorporation by schistosomes, but at the same time suggests that this inhibition is unlikely to represent the mechanism of schistosomicidal activity. In the present report we analyze the incorporation of radioactive precursors of DNA, RNA and proteins by sensitive, resistant and immature schistosomes in vitro and by HeLa cells. The effects of hycanthone and of the less mutagenic analog IA-4 [15] on these systems is studied in parallel with the effect of actinomycin D taken as a reference drug. Our results cannot be regarded as conclusive evidence for the hypothesis that hycanthone kills sehistosomes by blocking nucleic acid synthesis, nevertheless they permit to conclude that all the evidence presently available is fully compatible with that hypothesis. MATERIALS AND METHODS
Schistosomes. The origin and the maintenance of S. mansoni as well as the procedures used for animal infections have been described elsewhere [16]. A strain of S. mansoni y.~.,.a.i t m u ~ . v w a s r, m ua,__ a y supplied m b....... ,~j reslst,,,t to .~" ,!.___ ,_._ • 1973 by or. t~. tsueding (Johns Hopkins University, Baltimore, MD) and subsequently maintained in our laboratory. An additional resistant strain was isolated in our laboratory from a mouse treated with hycanthone 8 weeks after infection. The two resistant strains exhibited the same general characteristics and, for the purpose of the present experiments, they will be both referred to as 'resistant schistosomes'. HeLa cells ($3 clonal strain) were grown in suspension culture in Joklik modified Eagle's medium (Grand Island Biologi-~l Corporation) supplemented with 10% calf serum at a cell density between 1 × 10 s/ml and 5 × 10 s/ml. Cells were free of mycoplasma contamination as shown by the absence of labeled 16 S and 23 S ribosomal RNA [ 17]. Schistosome incubations. Adult schistosomes (46 days or more after infection), hycanthone-resistant schistosomes and immature worms ( 2 1 - 2 8 days old), obtained by perfusion of infected untreated mice, were suspended in Eagle's minimum essential medium (Dulbeeeo-modified) supplemented with 20% horse serum and were incubated at 37°C in a 5% CO2 atmosphere. Drugs were added at the indicated concentration to duplicate dishes containing worms of both sexes in 0.5 ml of medium, while identical duplicate
297 controls were left untreated. The number of worms/dish varied from 6 to 10 in different experiments, but the male/female ratio and the total number of worms were uniform in each experiment. 5-15 min after the addition of drugs, all dishes received the same amount of radioactive precursor, i.e. either 10/aCi/ml [5, 6-aH]uridine (45 Ci/mmol), 20 /aCi/ml [methyl-aH]thymidine (6.7 Ci/mmol), 20 #Ci/ml L-[4,5-3H]leucine (51.6 Ci/mmol), or 40/aCi/ml [5-aH]orotic acid (20 Ci/mmol), all purchased from New England Nuclear Corporation. At the end of the labeling period (1 h), worms were washed 3 times with cold salhle, resuspended in 1 ml water and disrupted by sonication. Sonicated samples were then precipitated with 5% trichloroaeetic acid (TCA), collected on glassfiber falters (Whatman GF/C), dipped 1 min in H202 in order to bleach the dark oigment contained in female worms, dried and counted in a liquid scintillation spectrometer. When [a H]orotic acid was used as a precursor, labeling in the 5 position prevented incorporation of radioactivity into DNA, as shown by DNAase resistance, RNAase sensitivity and alkaly sensitivity of the product. Samples labeled with [a H]leucine were treated, after sonication, with 0.5 N NaOH for 10 min at 37°C in order to hydrolyze radioactivity bound to tRNA and not incorporated into polypeptide chains. They were then neutralized and precipitated with TCA. Radioactivity of duplicate samples was usually within -+ 5% of their average, with a maximum of about + 15%. In order to determine the labeling of the total acid-soluble pool, aliquots of the sonicated samples were treated with 0.5 N perchloric acid (1 h at 4°C)and centrifuged for 10 min at 12 000 rev./min in a Sorvall SS34 rotor at 4°C. The acid-soluble radioactivity was then measured in the supernatant fraction. Alternatively, in the case of samples labeled with [aH]uridine or [a H] thymidine (where the acid-soluble radioactivity represents more than 90% of total counts), 100-#1 aliquots, out of 1 ml sonicaLed samples, were direcdy counted and used to estimate acid-soluble radioactivity. The rest of the sample was then TCA-precipitated and acid-insoluble radioactivity determined as described above. The synthetic activity of worms after drug removal was determined in samples treated for 3 0 - 4 5 min with either 15/~M hycanthone or 15/aM IA-4 or 3/aM actinomycin D, washed 3 times with fresh medium and reincubated at 37°C. At different times after drug removal, radioactive precursors were added and the incubation was carried out for 1 h as described above. In all the experiments reported here, the viability of worms was not detectably impaired at the end of incubations, as judged by the presence of active movements which, if anything, were slightly enhanced at hycanthone concentrations of about 10 taM.
HeLa cell incubations. 2 X l0 s cells in 0.5 ml medium were treated with drugs at the indicated concentrations and incubated with tritiated precursors in a 37°C shaking bath under the same conditions as described for worms. At the end of incubations, cells were washed and disrupted with 0.05% sodium dodecyl sulfate. Drugs. Hycanthone methanesulfonate was a gift from D~. A. Soria (Sterling Winthrop
298
Research Institute); IA-4 (a chloroindazole derivative of hycanthone) was a gift from Dr. J.F. de Serres (National Institute of Environmental Sciences); actinomycin D was purchased from Serva Co. RESULTS
Effect of different drug concentrations on adult sensitive schistosomes and HeLa cells. A dose-response study of the effect of hycanthone, IA-4 and actinomycin D on the incorporation of tritiated precursors into TCA-precipitable material was performed by incubating adult sensitive schistosomes and HeLa cells in the presence of different drug concentrations. The results are reported in Table I as percent inhibition of incorporation at the end of 1 h incubations and are calculated with respect to identical samples incubated in the absence of drugs. In adult schistosomes, hycanthone exerted its most pronounced effect on uridine incorporation, showing a substantial inhibition at concentrations which were of the same order of magnitude (i0/zM) as those required to kill schistosomes in rive [18] and in vitro [ 19]. Higher concentrations were required to produce a partial inhibition of thymidine and leucine incorporation. IA-4 showed effects which were very similar to those exerted by hycanthone. 3/zM actinomycin D (4/~g/ml) had comparable effects on uridine incorporation, while thymidine and leucine incorporations were practically unaffected. It is worth mentioning here that, in similar tests, the unrelated anti-schistosomal drug antimony potassium tartrate, at concentrations as high as 100/zM, had no effect on uridine incorporation by adult worms maintained in vitro (results not sho~vn). In HeLa cells, the most pronounced effects of hycanthone and IA-4 were apparent . . . . . . . k" q c o r p o r a t l"o n , ~'"" _agai_n o n Ilrldin,~ o u t , at variance ....... Wltil the results obtained with schistoseines, thymidine incorporation was also markedly inhibited, although to a lesse[ extent than uridine. 0.4/~g/ml actinomycin D (0.3/~M) gave the expected results with HeLa cells, i.e. a strong inhibition of uridine incorporation with practically no effect on thymidine and leucine incorporation. The experiments described in Table I we~'e carried out using a mixture of male and female schistosomes, usually with a slight predominance of male worms. The possible existence of sex-related differences was tested by incubating single-sex samples ( 6 - 8 worms) with tritiated precursors in the presence of 15 ~M hycan~hone. Female worms usually showed a lower sensitivity to hycanthone, a phenomer on which was observed upon labeling with uridine as well as with thymidine and leucine. In the course of these experiments, significant sex-related differences were also observed in control untreated samples. Each male schistosome wa~ able to incorporate more than twice the amount of [3H]urid';ne incorporated by each female, while female worms incorporated about 5 times the amount of [3 H] thymidine when cornpared to male worms. Male-female differences will be described in more detail in a separate report. Effect o f drugs on precursor uptake. Our analysis of schistosome syathetic activities is
299 based on the incorporation into TCA-precipitable material of radioactive precursors added to the medium. A drug-induced inhibition of incorporation observed under these conditions could be the result of either a block in the synthetic mechanism of each macromolecule, or the result of a block in file uptake of the external precursors into active cells, or both. The second possibility was tested by determining the amount of intracellular (intraschistosomal) acid-soluble radioactivity in the presence and in the absence of drugs, as described under Materials and Methods. At the end of our standard 1 h incubation, the uptake e f [3H]uridine was indeed inhibited by about 4.0% in the presence of 15/~M hycanthone. This result, however, cannot account for the 74% inhibition of acid-precipitable radioactivity observed under the same conditions (Table I). The uptake of thymidine and leucine was also decreased by hycanthone in a proportion corresponding to about one half the inhibition of TCAprecipitable counts. The effect of IA-4 was again very similar to the effect of hycanthone, while actinomycin D did not cause a significant inhibition of tritiated precursors uptake either in schistosomes or in HeLa cells. A more detailed study of the effects of hycanthone is shown in Fig. l, where the kinetics of uridine incorporation into acid-precipitable and acid-soluble material were determined under the continuous presence of the drug. The inhibition caused by hycanthone appeared very early and almost simultaneously for both the acid-soluble pool and the TCA-precipitable material, a fact which does not suggest a cause-effect relationship between the two phenomena. Contrasting with hycanthone effects, the results obtained with actinomycin D show an early block in the incorporation ot TCA-precipitable material, while thc precursor uptake into the acid-soluble pool is only slightly inhibited at later times (Fig. 1). A
!i
/
2 EE a,)
.=
EE
20
40
60
20
40
60
Time (min !
Fig. 1. Kinetics of labeling of the acid-soluble pool (A) and the acid-precipitable fraction (B) of adult schistosomes incubated with [aHluridine in the absence (controls, e) ~,nd in tl~.e presence of either 15 /~M hycanthone (o) or 3/~M actinomycin D (A). Drugs were added 5 rain before tiic radioactive plecvrscr. Eack. point represents the mean of duplicate samples.
~M
70 -+4 (2) 72 +-3 (2) 74+2(13) 81+-I (6) 84-+ 3 (2) 87 (I) 75+-4 (8) 31 ± 6 (2) 78 + 2 (6) (5)
77_+2
8 8 + 2 (4) 76 + 3 (3) 95 (I)
(1)
29
HeLa
0
(I)
31+7(4)
9 (I) 20 (I) 29+6(7) 41+-6(5) 50 (1)
[ s HI Thymidine Schistosomes
(I)
7 6 ± I (2) I0 (I)
66+I(3)
25
HeLa
18 _+2 (2) 0 (1) 12 +- 6 (3)
(1)
2~
(1) 34 ± 5 (5)
9
HeLa
28 ± 2 (3) 39 + 1 (3) 40 (1)
[ s H ] Leucine Schistosomes
could be estimated at I/~M for uridine, 40 uM for thymidine and 80 ~tM for leucme.
a Values represent the mean + S.E. of different experiments (No. of experiments in parenthesis) and are expressed as percent inhibition with respcct to the average incorporation of duplicate untreated controls in each experiment. b By extrapolation of the data contained in this table, the dose of hycanthone producing a 50% inhibition of precursor incorporation into schistosomes
5 I0 15 30 40 80 IA-4 15 Actinomycin D 0.3 3
Hycanthone b
Drug
[ 3H ] Uridine Schistosomes
Percent inhibition of incorporation of tritiated precursors into TCA-precipitable material of adult sensitive worms during 1 h incubations in the presence of various concentrations of drugs, a
TABLE 1
t,o
301
In an attempt to find conditions in which effects on nucleic acid synthesis could be studied separately from effects on precursor uptake, [3H]orotic acid was used in place of the more complex nudeozide uridine. Although the acid-precipitable radioactivity incorporated with [3H]orotic acid was only about one tenth of the value obtained using comparable amounts of [3H]uridine, the uptake of orotic acid into the acid-soluble intracellular pool was not inhibited by a concentration of 30/aM hycanthone (Table II). Under these conditions, an average 38% irreversible inhibition of [3H]orotic acid in corporation into adult schistosome RNA vas observed after I h incubation in the presence of the drug. Similar results were obtained with IA-4 (Table !l). In accordance with the results obtained using ['a H] uridine, these data permit to conclude that ~t least two phenomena coexist independently in hycanthone-treated schistosomes, i.e. an i~l[~Jbition of RNA synthetic mechanisms and a decreased uptake of certain precursor molecules. The latter phenomenon may be connected with membrane modifications induced by hycanthone, as suggested by the ability exhibited by this drug to protect human erythrocytes [20] significantly against hypotonic hemolysis (unpublished results).
Reversal of inhibition of uridine incorporation. Schistosomes and HeLa cells were incubated with either hycanthone, IA-4 or actinomycin D for 3 0 - 4 5 nun. Drugs were then removed by repeated washings alld the samples were resuspended in fresh medium for further incubation. At different times after drug removal the samples were exposed for 1 h to [ a H ] u r i d i n e as d e s c r i b e d u n d e r Materials and M e t h o d s . Fig. 2 s h o w s t h e in-
TABLE 1I Inhibition of incorporation of [3 H ] orotic acid into acid- soluble and acid-precipitable material of adult sensitive worms during 1 h incubation i~, thc presence of hycanthone or IA-4 and after removal of the drugs, a Percent inhibition of incorporation into Acid-soluble material In the presence of 30 uM hycanthone 2½ h after removal of hycanthone In the presence of 30 ~M IA-4 21/2 h after removal of IA-4
Acid-precipitable material
0 ± 1 (5)
38 ± 3 (5)
0
49
t 1)
16 ± 7 (4) 0
(1)
(1)
66 + 3 (4)
56
(1)
a Values represent the mean ± S.E. of different experiments (No. of experiments in parenthesis) and are expressed as percent inhibition with respect to the average incorporation of duplicate untreated controls in each experiment.
302
Sensitive
Immature
Resistant
HeLa
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o
I
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'
i
J
B lOOr
•
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g
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.,
•
:
I •2 3 4
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1234 ,.w+
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234
II:IIIL/VGI
Fig. 2. Inco..'poration of [3H]uridine, expressed as percent of incorporation in untreated controls, of samples exposed to the precursor for 1 h in the presence of drugs (time-zero points) or at different times after drug removal. Times reported on the abscissa refer to the end of the 1 h labeling period. Each point represents the mean of duplicate samples. To aid in visua_lizing the trend of each experiment, a line was drawn through two points: (i) a point on the vertical axis, corresponding to the mean of inhibitions in the presence of the drug; (ii) a point with the ordinate corresponding to the mean of all inhibition, s observed after drug removal, and with the abscissa corresponding to the mean of all times tested after drug removal. A, hycanthone (15 pM); B, IA-4 (15 pM); C, actinomycin D (3 pM for schistosomes; 0.3 pM for HeLa ceils).
corporation of [aH]uridine (expressed as percent of untreated controls) into TCAprecipitable material from worms and HeLa cells. The zero-time points were taken still in the presence of drugs. A profound inhibition of uridine incorporation in the presence of hycanthone was exhibited by aH the systems tested, including resistant worms - a result which is i:1 accordance with the observations of Neame et al. [ 14]. In the 4 h following the removal of hycanthone, it is evident that adult sensitive schistosomes did not resume normal levels of uridine incorporation: the inhibition appears to be irreversible (Fig. 2A). On the contrary, hycanthone resistant worms, immature worms
303
and laeLa cells, although starting from approximately the same level of inhibition as sensitive worms in the presence of hycanthone, exhibited an almost complete reversal of inhibition after removal of the drug. After exposure to IA-4 (Fig. 2B), sensitive worms showed an irreversible block of uridine incorporation, while HeLa cells promptly resumed activity upon washing of the drug, as observed with hycanthone. Immature worms, however, remained largely inhibited during the 4 h interval of our in vitro observation. Hycanthone-resistant worms treated with IA-4 showed a reduced inhibition of uridine incorporation in the presence of ~he drug (compare corr,esponding zero-time points of Fig. 2A and B) arid completely resumed activity upon removal of IA-4. Finally, the inhibition caused by actinomycin D (3/zM for schistosomes; 0.3/zM for HeLa cells) appeared to be irreversible in all the systems studied (Fig. 2C). The reversal of inhibition of ur~Jine incorporation was also studied using sensitive worms of a single sex. While male worms showed a behavior which was essentially identical to that described in Fig. 2, female worms exhibited a significant deviation from that pattern. Not only were female w.3rms less sensitive than males to inhibition in the presence of hycanthone (as previously noted), but they were also capable of a substantial reversal of inhibition during the 4 h period of incubation after drug removal (to be published). The inhibition of uridine uptake into acid-soluble material could always account for a fraction of the inhibition observed in acid-precipitable material and variations in the two parameters were usually parallel. Thus, immature and resistant worms, as well as HeLa cells, were able to revert uridine uptake inhibition after hycanthone removal, while the uptake appeared to be irreversibly blocked in sensitive worms (especially in males). In addition to the above experiments with [3 H] uridine, similar experiments were carried out using thymidine or leucine as radioactive precursors. In general, the initial inhibition of incorporation in the presence of hycanthone was not as pronounced as with uridine (see Table I), but sensitive worms showed a tendency toward a progressive increase of inhibition in the period subsequent to drug treatment and removal, while resistant worms, immature worms and HeLa cells pr~Jmptly reverted initial effects. While these phenomena were already noticeable at the relatively short times of our in vitro observations, a more complete picture of long-term events will be given in a subsequent report describing the effect of drugs after i~~.rive t~eatment. DISCUSSION
We have tested the hypotl~esis that hycanthone, a known DNA intercalating drug which inhibits nucleic acid synthesis in a number of systems, might exert its antischistosomal activity by interfering with the synthesis of macromolec:ules in S. mansoni. Our in vitro approach was aimed at the detection of drug-related biochemical differences between sensitive and resistant s,:histosomes, and between parasite and host tissues - the
304 latter being ~pre~ented by the human-derived HeLa cell cultures. We also analyzed the effect of the h?'c~mtho~e ~malog IA-4 which, being less mutagenic [ 15], could be supposed to intei, c~ ~:ifferently with nucleic acids [21]. The most c{:aspicuous effect of hycanthone and IA-4 was exerted on uridine incorpotation; while ~i~ywidine and leucine incorporations were inhibited to a smaller extent and at higher drug coneenttations (Table I). This observation suggests that RNA synthesis is most likely tc be a m,jor target of hycanthone activity. On the other hand, the inhibitions of DNA and protein synthesis, although of limited magnitude, appear very early after treatmep.t and may ~us represent 'primary' phenomena rather than a consequence of the decreased RNA synthesis. Therefore, the possibility should be left open tha~ a block in the synthesis of a particular DNA species (e.g. mi:'.-chondrial DNA) or a particular protein (e.g. ar~ enzyme) may indeed be crucial for schistosome death and yet represent only amino! fiaction o r the total synthetic activity. In addition, the finding that RNA synthesis is inhibited by hycanthone does not obviously exclude the existence of other possible drug targets in the schistosome. It should be noted that the alterations in the synthesis of macromolecules observed with hycanthone are not likely to be the consequence of a non-specific damage to worm physiology, since the unrelated schistosomicidal drug potassium antimony tartrate (which is thought to act by inhibiting schistosome phosphofructokinase) had no effect on worm macromolecular synthes~s. Our data show that incubation in the presence of hycanthone, IA-4 or actinomycin D, affects schistosomes in way similar to that described for various bacterial and mammalian systems [10-13] and conf'mned here for HeLa cells. Apart from some quantitative differences, thcse effects were equally present in sensitive and resi.~tant wo_.r.m..sof the two sexes and in immature schtstosomes (as well as in HeLa cells). The fact that resistant worms are affected recently led Neame et al. [ 14] to conclude that inhibition of nucleic acid synthesis is unlikely to represent ~he mechanism of action of hycanthone. We have reasoned that the condition which best mimics the in vivo activity of hycanthone is not its continuous contact with the worms, but rather a short pulse followed by a rapid dilution. We were led to this conclusion by considering that a single intramuscular dose is sufficient to exert a schistosomicidal effect [22] in spite of the fact that peak blood concentrations are reached 30 min after injection and are foUowed by a rapid decline with a plasma half-life of about 45 min [ 18, 23]. In addition, an in vitro contact as short as 15 min was sufficient to kill schistosomes which had been washed and transferred into untreated hamsters after drug exposure [19]. Based on these data, we analyzed the behavior of uridine incorporation in schistosomes which had been resuspended in drugfree medium after a 3 0 - 4 5 min contact with hycanthone. Sensitive 'wild-type' adult schistosomes presented an irreversible arrest of uridine incorporation, while resistant worms (like HeLa cells) promptly resumed normal levels of incorporation. The correlation between irreversibility of biochemical effects and parasite death was further supported by the finding that immature schistosomes, which are largely refractory to hycanthone killing [19, 24-26], showed a substantial reversal of the block of uridine
305
incorporation. In addition, female worms, which are known to be more resistant than males to hycanthone [19, 27, 28], showed lower susceptibility to and better recovery from the inhibition of uridine incorporation than male worms. IA-4 gave essentially the same results as hycanthone, except that uridine incorporation remained largely inhibited in immature worms treated with this drug. This result is consistent with our observation that mice treated with IA-4 on the 22nd day after infection showed a 38% reduction in the number of adult worms (unpublished results), and with the fact that Reid et al. [29] found a significant 'prophylactic' activity in IA-4 N-oxide administered to monkeys early after infection. As expected, the effect of actinomycin D was completely irreversible in all types of schistosomes as well as in HeLa cells. An additional observation which has emerged from this study is the fact that hycanthone and IA-4 significantly inhibited the uptake of some radioactive precursors (especially uridine) from the medium. This phenomenon, however, could not account for the largest part of the inhibition of uridine incorporation. A true action on RNA synthesis was demonstrated by the fact that the incorporation of orotic acid, a precursor whose uptake was not affected by hycanthone, was largely inhibited in the presence of the same drug. These results are in accordance with the hycanthone inhibition of DNAdependent RNA polymerase observed by Weinstein and Hirshberg [4] in cell-free systems. Our observation of a reduced entry of precursors into cells makes a precise assessment of the inhibition of RNA synthesis somewhat more complicate than previously thought [10, 11-13]. On the other hand, the phenomenon deserves further attention since it constitutes an indicator for another hycanthone target: cell membrane function. It is difficult to evaluate the possible consequences of an altered membrane permeability upon scb_istosome su_rviva!, but it should be pointed out that the inhibition of precursor uptake closely followed the inhibition of total incorporation and, just as the latter, it was promptly reversible in HeLa cells and resistant worms, while it was practically irreversible in sensitive worms. The main observations reported here for schistosomes kept in vitro have been confirmed using worms obtained from mice treated in vivo at various times before perfusion. Nucleic acid synthesis remained inhibited until death in sensitive schistosomes treated in vivo, while it resumed control values shortly after treatment of resistant and immature worms (manuscript in preparation). In conclusion, although several different mechanisms can be proposed to explain the schistosomicidal activity of hycanthone, our data suggest that the inhibition of RNA synthesis should be considered among the most important targets of the drug. ACKNOWLEDGEMENTS
This investigation was supported in part by a grant from Institut National de la Sant6 et de la Recherche M~dicale (contract A/FRC/FA/3690). The excellent technical assistance of Rolando Moroni is gratefully acknowledged. The authors appreciate the critical interest and helpful suggestions from Drs. L. Felicetti, P. Liberti and C. Vesco.
306 REFERENCES [ 1 ] Rosi, D., Peruzzotti, G., Dennis, E.W., Berberian, D.A., Freele, H. and Archer, S. (1965) A new, active metabolite of Miracil D. Nature (London) 208, 1005-1006. [2] Hiliman, G.R. and Senf~, A.W. (1975) Anticholinergic properties of the antischistosomal drug hycanthone. Am. J. Trop. Med. Hyg. 24, 827-834. [3] Tomosky-Sykes, TJC and Bueding, E. (1977) Effects of hycanthone on neuromuscular systems of Schistosoma mansonL J. Parasitol. 63, 259-266. [4] Weinstein, B. and Hirshberg, E. (1971) Mode of action of Miracil D. In: Progress in Molecular and Subcellular Biology, (Hahn, F~E., ed.), Vol. 2, pp. 232-246, Springer-Verlag, New York. [5] Waring, M. (1971) Binding of drugs to supercoiled circular DNA; evidence for and against intercalation. In: Progress in Molecular and Subcellular Biology (Hahn, F.E., ed.). Vol. 2, pp. 216231, Springer-Verlag, New York. [6] Haxtman, P.E., Levine, K., Hartman, Z. and Berger, H. (1971) Hycanthone: a frameshfft mutagen. Science 172, 1058-1060. [7] Moore, J.A. (1972) Teratogenicity of hycanthone in mice. Nature (London) 239, 107-109. [8] Haese, W.H., Smith, D.L. and Bueding, E. (1973) Hycanthone-induced hepatic changes in mice infected with Schistosoma mansonL J. PharmacoL Exp. Ther. 186, 430-439. [9] Yarinsky, A., Drobeck, HJ'., Freele, H., Wiland, J. and Gumaer, K.I. (1974) An 18-month study of parasitologic and tumorigenic effects of hycanthone in Schistosoma mansoni infected and non-infected mice. Toxicol. Appl. Pharmacol. 27, 169-182. [10] Weinstein, I.B., Carchman, R., Marner, E. and Hirshberg, E. (1967) Miracil D: effects on nucleic acid synthesis, protein synthesis and enzyme induction in Escherichia coll. Biochim. Biophys. Acta 142,440-449. [11] Bases, R. and Mendez, F. (1969) Reversible inhibition of ribosomal RNA synthesis in HeLa by lucant~hone .03VliracilD) with continued synthesis of DNA-like RNA. J. Cell. Physie~ 74, 283-294. [12] Wittner, M., Tanowitz, H. and Rosenbaum, M. (1971) Studies with the schistosomicide hycanthone. Inhibition of macromolecular synthesis and its reversal. Exp. Mol. Pathol. 14, 124-133. [13l Evffanova. O.I_ M~k~ro~, r'. ~: and At,,,I~a--A M.K (1975) A comparative study of the effects of lucanthone (Mixacil D) and actinomycin D on the Chinese hamster cells grown in cultures. J. Cell Physiol. 86, 261- 268. [ 14] Neame, K.D., Homewood, C.A., Marshall, I. and Jewsbury, J.M. (1978) The effect of hycanthone on nucleic acid synthesis by Schistosoma mansoni. Ann. Trop. Med. Parasitol. 72, 587-588. [15] Hulbert, P.B., Bueding, E. and Hartman, P.E. (1974) Hycanthone analogs: dissociation of mutagenic effects from antischistosomal effects. Science 186,647-648. [16] Cioli, D. (1976) Transfer of Schistosoma mansoni into the mesenteric veins of hamsters. Int. J. Parasitol. 6 , 3 4 9 - 3 5 4 . [17] Harley, EJ-l. and Rees, K.R. (1972) Mitochondrial RNA in mycoplasma infected HeLa cells. Biochin~. Biophys. Acta 259, 228-238. [18] Yarinsky, A., Hernandez, P. and Dennis, E.W. (1970) The uptake of tritiated hycanthone by male and female Schistosoma mansoni worms and distribution of the drug in plasma and whole blood of mice following a single intramuscular injection. Bull. WHO 42, 445-449. [19] Cioli, D. and Knopf, P.M. (1980) A study of the mode of action of hycanthone against Schistosoma mansoni in vivo and in vitro. Am, J. Trop. Med. Hyg. 29, 220- 226. [20] Seeman, P. and Weinstein, J. (1966) Erythrocyte membrane stabilization by tranquillizers and antihistamines. Biochem. Pharmacol. 15, 1737-1752. [21] Bueding, E., Elslager, E.F. and Jansma, W.B, (1975) A selective approach to the search for safer schistosomicidal drugs, l'roc, int. Conf. Schisto. 1975, 197-201. .
.
.
--.
. . . .
1,
-
-
-
~
..,....,it
.
.,-1.tJl~tal~.a~.,~,
.
307 [221 Berberian, D.A., Freele, H., Rosi, D., Dennis, E.W. and Archer, S. (1967) A comparison of oral and parenteral activity of hycanthone and lucanthone in experimental infections with Schistosoma mansoni. Am. J. Trop. Med. Hyg. 16,487-491. [23] Hernandez, P., Dennis, E.W. and Farah, A. (1971) Metabolism of the schistosomicidal agent hycanthone by rats and rhesus monkeys. Bull. WHO 45, 27-34. [24] Lapierre, J., Holler, C., Lebas-Saison, E., Le Prioux, B. and Roose, A. (1974) Action de i'hycanthone aux divers stades 6volutifs de l'infection exp~rimentale de la souris par Schistosoma mansoni. C.R. S~ances Soc. Biol. 168,452-454. [25] Pereira, L.H., Pellegrino, J. and Mello, R.T. (1975) Activity of known antischistosomal agents on early-developing forms of Schistosoma mansoni. J. Parasitol. 61,249-252. [26] Jansma, W.B., Rogers, S.H., Liu, C.L. and Bueding, E. (1977) Experimentally produced resistance ofSchistosoma mansoni to hycanthone. Am. J. Trop. Med. Hyg. 26, 926-936. [271 Foster, R., Cheetham, B.L., Mesmer, E.T. and King, D.F. (1970) Comparative studies of the action of mirasan, lucanthone, hycanthone and niridazole against Schistosoma mansoni in mice. Ann. Trop. Med. Parasitol. 65, 4 5 - 5 8 . [281 Lee, G. (1972) Aspects of the effect of thioxanthone on Schistosoma mansoni in mice and in vitro. Bull. WHO 46,397-402. I291 Reid, W.A., Loizeaux, P.S. and Reardon, M.J. (1977) The activity of a benzothiopyranoindazole (IA-4 N-oxide) against Schistosoma mansoni infection in rhesus monkeys. Am. J. Trop. Med. Hyg. 26,937-943.
295
© Elsevier/North-Holland Biomedical Press
EFFECT OF HYCANTHONE ON SCHISTOSOMA MANSONI MACROMOLECULAR SYNTHESIS IN VITRO
L ~ ' I A PICA ]dATTOCCIA, ALESSANDRA L E L I ' and DONATO CIOLI
Laboratory of Cell Biology, National Research Council, 18 Via Romagnosi, 00196 Rome, Italy (Received 14 April 1980; accepted 14 August 1980)
Adult, immature and hycanthone-resistant schistosomes were allowed to incorporate tritiated precursors of maeromolecule ~ynthesis in vitro, either in the presence of various concentrations of hycanthone, o~" at various times after removal of the drug. The effect on worms was compared to that on Hel.a cells. The results show that hyeanthone markedly inhibited the incorporation of uridine in all the sy ,terns studied, while the incorporation of thymidine and leucine was only secondarily affected. TI~c inhibition of uridine incorporation reflected in part a decreased uptake of the radioactive precursor. ' f ' z hycanthone-induced inhibition of uridine incorporation was essentially irreversible upon remo :~t[ of the dxug ill adult sehistosomes, while it was completely reversible in hycanthoneresistant wol.as, in immature worms and in HeLa cells. The effects of a hycanthone analog, IA-4, were largely comparable to the effects of the parent compound. These results suggest that the inhibition of RNA synthesis can be a possible explanation for the mechanism of the schistosomieidal action of hycantho~e. Key words: Hycanthone, Hycanthone analog IA-4, RNA synthesis, Drug mechanism, In vitro labeling,
Schistosoma mansoni.
INTRODUCTION
Hycanthone is one of the most widely vsed drugs in human infections with Schistosoma ma,2soni and S. hematobium. It is one of the metabolites of a previously used compound, lucanthone or Miracil D [ 1 ]. The mechanism of its schistosomicidal activity is not dear, although hypotheses have been advanced on its possible interference with the functioning of neurotransmitters in the worms [2, 3 ]. The hvcanthone molecule contains a planar aromatic triple ring system which can interact w~th DNA [4] probably by intercalating between adjacent base pairs [5]. Presumably connected with these biochemical properties, hycanthone is mutagenic [6], teratogenlic [7] and possibly carcinogenic [8, 9]. The effects of hycanthone and lucan-
~bbreviatic,ns: TCA, trich!oroacetic acid; IA-4, 8-ch!oro-2[2-(diethylarnino) ethyll-2H-[ 1 ]benzo~iopyrano.[4, 3, 2-cd ]..indazole-5-methanesulfonate.
296
thone on nucleic acid and protein synthesis have been studied in a number of bacterial and mammalian systems [10-13]. In general, a pronounced but reversible inhibition of RNA synthesis was observed, while DNA and protein synthesis were affected to a lesser and more variable extent. On the basis of the above-mentioned data, the possibility should be considered that the schistosomicidal action of hycanthone might be due to its interference with the macromoleeular synthesis of the parasite. Surprisingly, however, no direct studies were available, until recently, on hycanthone effects on schistosome nucleic acid and protein synthesis. A recent communication [ 14] actually reports an inhibition of [3H]adenosine incorporation by schistosomes, but at the same time suggests that this inhibition is unlikely to represent the mechanism of schistosomicidal activity. In the present report we analyze the incorporation of radioactive precursors of DNA, RNA and proteins by sensitive, resistant and immature schistosomes in vitro and by HeLa cells. The effects of hycanthone and of the less mutagenic analog IA-4 [15] on these systems is studied in parallel with the effect of actinomycin D taken as a reference drug. Our results cannot be regarded as conclusive evidence for the hypothesis that hycanthone kills sehistosomes by blocking nucleic acid synthesis, nevertheless they permit to conclude that all the evidence presently available is fully compatible with that hypothesis. MATERIALS AND METHODS
Schistosomes. The origin and the maintenance of S. mansoni as well as the procedures used for animal infections have been described elsewhere [16]. A strain of S. mansoni y.~.,.a.i t m u ~ . v w a s r, m ua,__ a y supplied m b....... ,~j reslst,,,t to .~" ,!.___ ,_._ • 1973 by or. t~. tsueding (Johns Hopkins University, Baltimore, MD) and subsequently maintained in our laboratory. An additional resistant strain was isolated in our laboratory from a mouse treated with hycanthone 8 weeks after infection. The two resistant strains exhibited the same general characteristics and, for the purpose of the present experiments, they will be both referred to as 'resistant schistosomes'. HeLa cells ($3 clonal strain) were grown in suspension culture in Joklik modified Eagle's medium (Grand Island Biologi-~l Corporation) supplemented with 10% calf serum at a cell density between 1 × 10 s/ml and 5 × 10 s/ml. Cells were free of mycoplasma contamination as shown by the absence of labeled 16 S and 23 S ribosomal RNA [ 17]. Schistosome incubations. Adult schistosomes (46 days or more after infection), hycanthone-resistant schistosomes and immature worms ( 2 1 - 2 8 days old), obtained by perfusion of infected untreated mice, were suspended in Eagle's minimum essential medium (Dulbeeeo-modified) supplemented with 20% horse serum and were incubated at 37°C in a 5% CO2 atmosphere. Drugs were added at the indicated concentration to duplicate dishes containing worms of both sexes in 0.5 ml of medium, while identical duplicate
297 controls were left untreated. The number of worms/dish varied from 6 to 10 in different experiments, but the male/female ratio and the total number of worms were uniform in each experiment. 5-15 min after the addition of drugs, all dishes received the same amount of radioactive precursor, i.e. either 10/aCi/ml [5, 6-aH]uridine (45 Ci/mmol), 20 /aCi/ml [methyl-aH]thymidine (6.7 Ci/mmol), 20 #Ci/ml L-[4,5-3H]leucine (51.6 Ci/mmol), or 40/aCi/ml [5-aH]orotic acid (20 Ci/mmol), all purchased from New England Nuclear Corporation. At the end of the labeling period (1 h), worms were washed 3 times with cold salhle, resuspended in 1 ml water and disrupted by sonication. Sonicated samples were then precipitated with 5% trichloroaeetic acid (TCA), collected on glassfiber falters (Whatman GF/C), dipped 1 min in H202 in order to bleach the dark oigment contained in female worms, dried and counted in a liquid scintillation spectrometer. When [a H]orotic acid was used as a precursor, labeling in the 5 position prevented incorporation of radioactivity into DNA, as shown by DNAase resistance, RNAase sensitivity and alkaly sensitivity of the product. Samples labeled with [a H]leucine were treated, after sonication, with 0.5 N NaOH for 10 min at 37°C in order to hydrolyze radioactivity bound to tRNA and not incorporated into polypeptide chains. They were then neutralized and precipitated with TCA. Radioactivity of duplicate samples was usually within -+ 5% of their average, with a maximum of about + 15%. In order to determine the labeling of the total acid-soluble pool, aliquots of the sonicated samples were treated with 0.5 N perchloric acid (1 h at 4°C)and centrifuged for 10 min at 12 000 rev./min in a Sorvall SS34 rotor at 4°C. The acid-soluble radioactivity was then measured in the supernatant fraction. Alternatively, in the case of samples labeled with [aH]uridine or [a H] thymidine (where the acid-soluble radioactivity represents more than 90% of total counts), 100-#1 aliquots, out of 1 ml sonicaLed samples, were direcdy counted and used to estimate acid-soluble radioactivity. The rest of the sample was then TCA-precipitated and acid-insoluble radioactivity determined as described above. The synthetic activity of worms after drug removal was determined in samples treated for 3 0 - 4 5 min with either 15/~M hycanthone or 15/aM IA-4 or 3/aM actinomycin D, washed 3 times with fresh medium and reincubated at 37°C. At different times after drug removal, radioactive precursors were added and the incubation was carried out for 1 h as described above. In all the experiments reported here, the viability of worms was not detectably impaired at the end of incubations, as judged by the presence of active movements which, if anything, were slightly enhanced at hycanthone concentrations of about 10 taM.
HeLa cell incubations. 2 X l0 s cells in 0.5 ml medium were treated with drugs at the indicated concentrations and incubated with tritiated precursors in a 37°C shaking bath under the same conditions as described for worms. At the end of incubations, cells were washed and disrupted with 0.05% sodium dodecyl sulfate. Drugs. Hycanthone methanesulfonate was a gift from D~. A. Soria (Sterling Winthrop
298
Research Institute); IA-4 (a chloroindazole derivative of hycanthone) was a gift from Dr. J.F. de Serres (National Institute of Environmental Sciences); actinomycin D was purchased from Serva Co. RESULTS
Effect of different drug concentrations on adult sensitive schistosomes and HeLa cells. A dose-response study of the effect of hycanthone, IA-4 and actinomycin D on the incorporation of tritiated precursors into TCA-precipitable material was performed by incubating adult sensitive schistosomes and HeLa cells in the presence of different drug concentrations. The results are reported in Table I as percent inhibition of incorporation at the end of 1 h incubations and are calculated with respect to identical samples incubated in the absence of drugs. In adult schistosomes, hycanthone exerted its most pronounced effect on uridine incorporation, showing a substantial inhibition at concentrations which were of the same order of magnitude (i0/zM) as those required to kill schistosomes in rive [18] and in vitro [ 19]. Higher concentrations were required to produce a partial inhibition of thymidine and leucine incorporation. IA-4 showed effects which were very similar to those exerted by hycanthone. 3/zM actinomycin D (4/~g/ml) had comparable effects on uridine incorporation, while thymidine and leucine incorporations were practically unaffected. It is worth mentioning here that, in similar tests, the unrelated anti-schistosomal drug antimony potassium tartrate, at concentrations as high as 100/zM, had no effect on uridine incorporation by adult worms maintained in vitro (results not sho~vn). In HeLa cells, the most pronounced effects of hycanthone and IA-4 were apparent . . . . . . . k" q c o r p o r a t l"o n , ~'"" _agai_n o n Ilrldin,~ o u t , at variance ....... Wltil the results obtained with schistoseines, thymidine incorporation was also markedly inhibited, although to a lesse[ extent than uridine. 0.4/~g/ml actinomycin D (0.3/~M) gave the expected results with HeLa cells, i.e. a strong inhibition of uridine incorporation with practically no effect on thymidine and leucine incorporation. The experiments described in Table I we~'e carried out using a mixture of male and female schistosomes, usually with a slight predominance of male worms. The possible existence of sex-related differences was tested by incubating single-sex samples ( 6 - 8 worms) with tritiated precursors in the presence of 15 ~M hycan~hone. Female worms usually showed a lower sensitivity to hycanthone, a phenomer on which was observed upon labeling with uridine as well as with thymidine and leucine. In the course of these experiments, significant sex-related differences were also observed in control untreated samples. Each male schistosome wa~ able to incorporate more than twice the amount of [3H]urid';ne incorporated by each female, while female worms incorporated about 5 times the amount of [3 H] thymidine when cornpared to male worms. Male-female differences will be described in more detail in a separate report. Effect o f drugs on precursor uptake. Our analysis of schistosome syathetic activities is
299 based on the incorporation into TCA-precipitable material of radioactive precursors added to the medium. A drug-induced inhibition of incorporation observed under these conditions could be the result of either a block in the synthetic mechanism of each macromolecule, or the result of a block in file uptake of the external precursors into active cells, or both. The second possibility was tested by determining the amount of intracellular (intraschistosomal) acid-soluble radioactivity in the presence and in the absence of drugs, as described under Materials and Methods. At the end of our standard 1 h incubation, the uptake e f [3H]uridine was indeed inhibited by about 4.0% in the presence of 15/~M hycanthone. This result, however, cannot account for the 74% inhibition of acid-precipitable radioactivity observed under the same conditions (Table I). The uptake of thymidine and leucine was also decreased by hycanthone in a proportion corresponding to about one half the inhibition of TCAprecipitable counts. The effect of IA-4 was again very similar to the effect of hycanthone, while actinomycin D did not cause a significant inhibition of tritiated precursors uptake either in schistosomes or in HeLa cells. A more detailed study of the effects of hycanthone is shown in Fig. l, where the kinetics of uridine incorporation into acid-precipitable and acid-soluble material were determined under the continuous presence of the drug. The inhibition caused by hycanthone appeared very early and almost simultaneously for both the acid-soluble pool and the TCA-precipitable material, a fact which does not suggest a cause-effect relationship between the two phenomena. Contrasting with hycanthone effects, the results obtained with actinomycin D show an early block in the incorporation ot TCA-precipitable material, while thc precursor uptake into the acid-soluble pool is only slightly inhibited at later times (Fig. 1). A
!i
/
2 EE a,)
.=
EE
20
40
60
20
40
60
Time (min !
Fig. 1. Kinetics of labeling of the acid-soluble pool (A) and the acid-precipitable fraction (B) of adult schistosomes incubated with [aHluridine in the absence (controls, e) ~,nd in tl~.e presence of either 15 /~M hycanthone (o) or 3/~M actinomycin D (A). Drugs were added 5 rain before tiic radioactive plecvrscr. Eack. point represents the mean of duplicate samples.
~M
70 -+4 (2) 72 +-3 (2) 74+2(13) 81+-I (6) 84-+ 3 (2) 87 (I) 75+-4 (8) 31 ± 6 (2) 78 + 2 (6) (5)
77_+2
8 8 + 2 (4) 76 + 3 (3) 95 (I)
(1)
29
HeLa
0
(I)
31+7(4)
9 (I) 20 (I) 29+6(7) 41+-6(5) 50 (1)
[ s HI Thymidine Schistosomes
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66+I(3)
25
HeLa
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(1)
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9
HeLa
28 ± 2 (3) 39 + 1 (3) 40 (1)
[ s H ] Leucine Schistosomes
could be estimated at I/~M for uridine, 40 uM for thymidine and 80 ~tM for leucme.
a Values represent the mean + S.E. of different experiments (No. of experiments in parenthesis) and are expressed as percent inhibition with respcct to the average incorporation of duplicate untreated controls in each experiment. b By extrapolation of the data contained in this table, the dose of hycanthone producing a 50% inhibition of precursor incorporation into schistosomes
5 I0 15 30 40 80 IA-4 15 Actinomycin D 0.3 3
Hycanthone b
Drug
[ 3H ] Uridine Schistosomes
Percent inhibition of incorporation of tritiated precursors into TCA-precipitable material of adult sensitive worms during 1 h incubations in the presence of various concentrations of drugs, a
TABLE 1
t,o
301
In an attempt to find conditions in which effects on nucleic acid synthesis could be studied separately from effects on precursor uptake, [3H]orotic acid was used in place of the more complex nudeozide uridine. Although the acid-precipitable radioactivity incorporated with [3H]orotic acid was only about one tenth of the value obtained using comparable amounts of [3H]uridine, the uptake of orotic acid into the acid-soluble intracellular pool was not inhibited by a concentration of 30/aM hycanthone (Table II). Under these conditions, an average 38% irreversible inhibition of [3H]orotic acid in corporation into adult schistosome RNA vas observed after I h incubation in the presence of the drug. Similar results were obtained with IA-4 (Table !l). In accordance with the results obtained using ['a H] uridine, these data permit to conclude that ~t least two phenomena coexist independently in hycanthone-treated schistosomes, i.e. an i~l[~Jbition of RNA synthetic mechanisms and a decreased uptake of certain precursor molecules. The latter phenomenon may be connected with membrane modifications induced by hycanthone, as suggested by the ability exhibited by this drug to protect human erythrocytes [20] significantly against hypotonic hemolysis (unpublished results).
Reversal of inhibition of uridine incorporation. Schistosomes and HeLa cells were incubated with either hycanthone, IA-4 or actinomycin D for 3 0 - 4 5 nun. Drugs were then removed by repeated washings alld the samples were resuspended in fresh medium for further incubation. At different times after drug removal the samples were exposed for 1 h to [ a H ] u r i d i n e as d e s c r i b e d u n d e r Materials and M e t h o d s . Fig. 2 s h o w s t h e in-
TABLE 1I Inhibition of incorporation of [3 H ] orotic acid into acid- soluble and acid-precipitable material of adult sensitive worms during 1 h incubation i~, thc presence of hycanthone or IA-4 and after removal of the drugs, a Percent inhibition of incorporation into Acid-soluble material In the presence of 30 uM hycanthone 2½ h after removal of hycanthone In the presence of 30 ~M IA-4 21/2 h after removal of IA-4
Acid-precipitable material
0 ± 1 (5)
38 ± 3 (5)
0
49
t 1)
16 ± 7 (4) 0
(1)
(1)
66 + 3 (4)
56
(1)
a Values represent the mean ± S.E. of different experiments (No. of experiments in parenthesis) and are expressed as percent inhibition with respect to the average incorporation of duplicate untreated controls in each experiment.
302
Sensitive
Immature
Resistant
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i
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Fig. 2. Inco..'poration of [3H]uridine, expressed as percent of incorporation in untreated controls, of samples exposed to the precursor for 1 h in the presence of drugs (time-zero points) or at different times after drug removal. Times reported on the abscissa refer to the end of the 1 h labeling period. Each point represents the mean of duplicate samples. To aid in visua_lizing the trend of each experiment, a line was drawn through two points: (i) a point on the vertical axis, corresponding to the mean of inhibitions in the presence of the drug; (ii) a point with the ordinate corresponding to the mean of all inhibition, s observed after drug removal, and with the abscissa corresponding to the mean of all times tested after drug removal. A, hycanthone (15 pM); B, IA-4 (15 pM); C, actinomycin D (3 pM for schistosomes; 0.3 pM for HeLa ceils).
corporation of [aH]uridine (expressed as percent of untreated controls) into TCAprecipitable material from worms and HeLa cells. The zero-time points were taken still in the presence of drugs. A profound inhibition of uridine incorporation in the presence of hycanthone was exhibited by aH the systems tested, including resistant worms - a result which is i:1 accordance with the observations of Neame et al. [ 14]. In the 4 h following the removal of hycanthone, it is evident that adult sensitive schistosomes did not resume normal levels of uridine incorporation: the inhibition appears to be irreversible (Fig. 2A). On the contrary, hycanthone resistant worms, immature worms
303
and laeLa cells, although starting from approximately the same level of inhibition as sensitive worms in the presence of hycanthone, exhibited an almost complete reversal of inhibition after removal of the drug. After exposure to IA-4 (Fig. 2B), sensitive worms showed an irreversible block of uridine incorporation, while HeLa cells promptly resumed activity upon washing of the drug, as observed with hycanthone. Immature worms, however, remained largely inhibited during the 4 h interval of our in vitro observation. Hycanthone-resistant worms treated with IA-4 showed a reduced inhibition of uridine incorporation in the presence of ~he drug (compare corr,esponding zero-time points of Fig. 2A and B) arid completely resumed activity upon removal of IA-4. Finally, the inhibition caused by actinomycin D (3/zM for schistosomes; 0.3/zM for HeLa cells) appeared to be irreversible in all the systems studied (Fig. 2C). The reversal of inhibition of ur~Jine incorporation was also studied using sensitive worms of a single sex. While male worms showed a behavior which was essentially identical to that described in Fig. 2, female worms exhibited a significant deviation from that pattern. Not only were female w.3rms less sensitive than males to inhibition in the presence of hycanthone (as previously noted), but they were also capable of a substantial reversal of inhibition during the 4 h period of incubation after drug removal (to be published). The inhibition of uridine uptake into acid-soluble material could always account for a fraction of the inhibition observed in acid-precipitable material and variations in the two parameters were usually parallel. Thus, immature and resistant worms, as well as HeLa cells, were able to revert uridine uptake inhibition after hycanthone removal, while the uptake appeared to be irreversibly blocked in sensitive worms (especially in males). In addition to the above experiments with [3 H] uridine, similar experiments were carried out using thymidine or leucine as radioactive precursors. In general, the initial inhibition of incorporation in the presence of hycanthone was not as pronounced as with uridine (see Table I), but sensitive worms showed a tendency toward a progressive increase of inhibition in the period subsequent to drug treatment and removal, while resistant worms, immature worms and HeLa cells pr~Jmptly reverted initial effects. While these phenomena were already noticeable at the relatively short times of our in vitro observations, a more complete picture of long-term events will be given in a subsequent report describing the effect of drugs after i~~.rive t~eatment. DISCUSSION
We have tested the hypotl~esis that hycanthone, a known DNA intercalating drug which inhibits nucleic acid synthesis in a number of systems, might exert its antischistosomal activity by interfering with the synthesis of macromolec:ules in S. mansoni. Our in vitro approach was aimed at the detection of drug-related biochemical differences between sensitive and resistant s,:histosomes, and between parasite and host tissues - the
304 latter being ~pre~ented by the human-derived HeLa cell cultures. We also analyzed the effect of the h?'c~mtho~e ~malog IA-4 which, being less mutagenic [ 15], could be supposed to intei, c~ ~:ifferently with nucleic acids [21]. The most c{:aspicuous effect of hycanthone and IA-4 was exerted on uridine incorpotation; while ~i~ywidine and leucine incorporations were inhibited to a smaller extent and at higher drug coneenttations (Table I). This observation suggests that RNA synthesis is most likely tc be a m,jor target of hycanthone activity. On the other hand, the inhibitions of DNA and protein synthesis, although of limited magnitude, appear very early after treatmep.t and may ~us represent 'primary' phenomena rather than a consequence of the decreased RNA synthesis. Therefore, the possibility should be left open tha~ a block in the synthesis of a particular DNA species (e.g. mi:'.-chondrial DNA) or a particular protein (e.g. ar~ enzyme) may indeed be crucial for schistosome death and yet represent only amino! fiaction o r the total synthetic activity. In addition, the finding that RNA synthesis is inhibited by hycanthone does not obviously exclude the existence of other possible drug targets in the schistosome. It should be noted that the alterations in the synthesis of macromolecules observed with hycanthone are not likely to be the consequence of a non-specific damage to worm physiology, since the unrelated schistosomicidal drug potassium antimony tartrate (which is thought to act by inhibiting schistosome phosphofructokinase) had no effect on worm macromolecular synthes~s. Our data show that incubation in the presence of hycanthone, IA-4 or actinomycin D, affects schistosomes in way similar to that described for various bacterial and mammalian systems [10-13] and conf'mned here for HeLa cells. Apart from some quantitative differences, thcse effects were equally present in sensitive and resi.~tant wo_.r.m..sof the two sexes and in immature schtstosomes (as well as in HeLa cells). The fact that resistant worms are affected recently led Neame et al. [ 14] to conclude that inhibition of nucleic acid synthesis is unlikely to represent ~he mechanism of action of hycanthone. We have reasoned that the condition which best mimics the in vivo activity of hycanthone is not its continuous contact with the worms, but rather a short pulse followed by a rapid dilution. We were led to this conclusion by considering that a single intramuscular dose is sufficient to exert a schistosomicidal effect [22] in spite of the fact that peak blood concentrations are reached 30 min after injection and are foUowed by a rapid decline with a plasma half-life of about 45 min [ 18, 23]. In addition, an in vitro contact as short as 15 min was sufficient to kill schistosomes which had been washed and transferred into untreated hamsters after drug exposure [19]. Based on these data, we analyzed the behavior of uridine incorporation in schistosomes which had been resuspended in drugfree medium after a 3 0 - 4 5 min contact with hycanthone. Sensitive 'wild-type' adult schistosomes presented an irreversible arrest of uridine incorporation, while resistant worms (like HeLa cells) promptly resumed normal levels of incorporation. The correlation between irreversibility of biochemical effects and parasite death was further supported by the finding that immature schistosomes, which are largely refractory to hycanthone killing [19, 24-26], showed a substantial reversal of the block of uridine
305
incorporation. In addition, female worms, which are known to be more resistant than males to hycanthone [19, 27, 28], showed lower susceptibility to and better recovery from the inhibition of uridine incorporation than male worms. IA-4 gave essentially the same results as hycanthone, except that uridine incorporation remained largely inhibited in immature worms treated with this drug. This result is consistent with our observation that mice treated with IA-4 on the 22nd day after infection showed a 38% reduction in the number of adult worms (unpublished results), and with the fact that Reid et al. [29] found a significant 'prophylactic' activity in IA-4 N-oxide administered to monkeys early after infection. As expected, the effect of actinomycin D was completely irreversible in all types of schistosomes as well as in HeLa cells. An additional observation which has emerged from this study is the fact that hycanthone and IA-4 significantly inhibited the uptake of some radioactive precursors (especially uridine) from the medium. This phenomenon, however, could not account for the largest part of the inhibition of uridine incorporation. A true action on RNA synthesis was demonstrated by the fact that the incorporation of orotic acid, a precursor whose uptake was not affected by hycanthone, was largely inhibited in the presence of the same drug. These results are in accordance with the hycanthone inhibition of DNAdependent RNA polymerase observed by Weinstein and Hirshberg [4] in cell-free systems. Our observation of a reduced entry of precursors into cells makes a precise assessment of the inhibition of RNA synthesis somewhat more complicate than previously thought [10, 11-13]. On the other hand, the phenomenon deserves further attention since it constitutes an indicator for another hycanthone target: cell membrane function. It is difficult to evaluate the possible consequences of an altered membrane permeability upon scb_istosome su_rviva!, but it should be pointed out that the inhibition of precursor uptake closely followed the inhibition of total incorporation and, just as the latter, it was promptly reversible in HeLa cells and resistant worms, while it was practically irreversible in sensitive worms. The main observations reported here for schistosomes kept in vitro have been confirmed using worms obtained from mice treated in vivo at various times before perfusion. Nucleic acid synthesis remained inhibited until death in sensitive schistosomes treated in vivo, while it resumed control values shortly after treatment of resistant and immature worms (manuscript in preparation). In conclusion, although several different mechanisms can be proposed to explain the schistosomicidal activity of hycanthone, our data suggest that the inhibition of RNA synthesis should be considered among the most important targets of the drug. ACKNOWLEDGEMENTS
This investigation was supported in part by a grant from Institut National de la Sant6 et de la Recherche M~dicale (contract A/FRC/FA/3690). The excellent technical assistance of Rolando Moroni is gratefully acknowledged. The authors appreciate the critical interest and helpful suggestions from Drs. L. Felicetti, P. Liberti and C. Vesco.
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