Trypanosoma (Schizotrypanum) dionisii as a model for chemotherapeutic studies related to Chagas's disease

80 TRANSACTIONS

OP THE

Trypanosoma

ROYALSOCIETY OPTROPICAL

MEDICINE

AND HYGIENE,

VOL.. 75, No.

1, 1981

dionisii as a model for chemotherapeutic studies related to Chagas’s disease

(Schizotrypanum)

J. R. BAKER* AND LINDA Medical

Research Council Biochemical

F. SELDEN Parasitology Unit, Molten0 Institute, CB2 3EE, UK.

Summary Cultivated trypomastigotes of Trypanosoma (Schizotrypanum) dionisii were added to cultures of buffalo lung (BL) cells at 37°C in a ratio of 500 parasites per cell. More than 60% of the cells became infected with amastigotes and the system was used to test trypanosomicidal activity and cytotoxicity of possible chemotherapeutic agents for use in Chagas’s disease. Three nitroheterocyclic compounds known to be active against T. cruzi in cell cultures (nifurtimox, benznidazole and SQ 18506) were active at similar levels against intracellular T. dionisii (1. 10 and 0.1 ue: ml-l resoectively); concentratib& of 10, 10 ‘akd 1 pg m&l respectively were cytotoxic. Other substances, not regarded as active against T. cruzi (metronidazole, pentamidine and pentostam), had little or no trypanosomicidal effect even at 100 pg ml-l. A recently developed nitroheterocyclic compound (MK 436) was active. and not cvtotoxic. at 10 ue r&l and’ possibly warrants furlher in;estigatibG Tetraethylthiuram disulphide, an inhibitor of threonine dehydrogenase which prevents growth of extracellular T. cruzi and T. dionisii in vitro at to 1 pi% ml-l, was only slightly trypanosomicidal intracellular T. dionisii at this dose; it was also somewhat cytotoxic. Introduction Trypanosoma cruzi causes human Chagas’s disease, for which there is no adequate chemotherapy (GUTTERIDGE, 1976; FIFE, 1977), in South and Central America. BAYLES et al. (1966) and GUTTERIDGE et al. (1969, 1978b) investigated the therapeutic potential of various compounds in vitro in cell cultures infected with T. cruzi. and GUTTERIDGE et al. (1978a) used mice infected* with the parasite as a simple primary “screen” for such compounds in viva. All such systems however involve the risk of accidental infection of personnel and it has been suggested that T. dionisii, a member of the same subgenus (Schizotrypanum) as T. cruzi for which no evidence exists of human infectivity, could serve as a safer test organism (BAKER, 1976). T. dionisii grown axenically in vitro has similar sensitivities to several compounds as does T. cruzi (GABORAK et al., 1977), and this paper evaluates its use in mammalian cell cultures at 37°C for testing potentially trypanosomicidal substances. * Present address (for reprint requests): The Culture Centre of Algae and Protozoa (Natural Environment Research Council), 36 Storey’s Way, Cambridge, CB3 ODT, England.

Downing

Street, Cambridge,

Materials and Methods Trypunosomes Stock P2 of Trypunosomu dionisii was isolated from Piaistrellus aibistrellus in 1971 (nrimarv isolate nimber LOkfiON/71/BPUC/2; ~~
81

J. R. BAKER AND L. F. SELDEN

I I zg .t? i CACA

three (occasionally four) days (i.e., seven or eight days after their initiation), all cultures were washed with medium 199 without serum, fixed for 30 min in Bouin’s fluid, washed with 70% ethanol and stained by the Giemsa-colophonium method (BRAY & GARNHAM, 1962) as described by BAKER et al. (1972). Slides were coded so that their treatment was unknown to the enumerator, and examined using a /: 10 square-grid ocular and x 50 oil immersion objective on a Wild M20 microscope. Counts of all cells, cells infected with one to ten parasites and cells containing more than 10 parasites were made on 10 (or, occasionally, more) microscope fields at sites distributed evenly over the surface of each stained monolayer (a circle about 1 cm in diameter) at predetermined settings of the mechanical stage. Replicate (usually triplicate) cultures were examined and means with their standard errors (SEM) calculated (BAKER & LISTON, 1978). The difference between two means was assumed to be significant only if it exceeded the sum of twice their SEMs. Extracellular trypanosomes in supernatant medium from cell cultures were counted with the aid of an improved Neubauer haemocytometer. Results

1. Reproducibility of resuIts within experiments In one experiment using BESM cells and SQ 18506, duplicate counts were made of cells in, firstly, 30 microscope fields per slide and, secondly, 10 fields only. There was no significant difference between the two counts (Fig. 1). 2. Reproducibility between experiments Two to four separate experiments were performed at different times using different cell and parasite inocula (Table II). The only results differing significantly from others obtained with the same drugs and concentrations were the second experiments in the groups using nifurtimox at 1 pg ml-l and SQ 18506 at 0.1 pg ml-l. 3. Trypanosomicidal effect of “standard” nitroheterocyclic compounds Nifurtimox, benznidazole and SQ 18506 at 10 pg ml-l all showed marked trypanosomicidal properties. At 1 pg ml-l, only nifurtimox and SQ 18506 were trypanosomicidal (Table II). 4. Activity threshold of “standard” nitroheterocyclic compounds One experiment using BESM cells and SQ 18506 at three serial tenfold dilutions has been reported in section 1 (Fig. 1). Similar experiments with BL cells showed that the trypanosomicidal effects of SQ 18506 and nifurtimox were diluted out, with effective thresholds around 0.1 and 1 pg ml-l (Table II). 5. Cytotoxicity of “standard” nitroheterocyclic compounds Preliminary trials (section 4) showed that 10 pg ml-l of nifurtimox, benznidazole or SQ 18506 were markedly cytotoxic to BL cells. At 1 pg ml-l only SQ 18506 showed this effect, and at lower

82

T. &onisii

AS A DRUG

TEST

MODEL

FOR

T. C?WZi

Figs. l-4. Trypanosomicidal and cytotoxic effects of different compounds on 1 ml cultures of BESM (Fig. 1) or BL (Figs. 2-4) cells initiated on day 0, infected with T. dionisii cm day 1, treated on day 4 and examined on day 7 (day 8 in Fig. 2). Horizontal bars represent means (n = 3), vertical bars represent f 2 SEM. Drug concentrations in pg ml“; zero concentration = treatment with appropriate solvent (0.1 X) only (see Table 1). Column 1 = total cell count in 10 microscope fields, x 500 (30 fields in Fig. la); column 2 = percentage of cells infected with T. dionisii; column 3 = percentage of infected cells containing more than 10 parasites. Horizontal dashed lines represent control mean minus 2 SEM; values below this are deemed significant.

Fig. 3. Effects of MK 436 (MK) two experiments (i and ii‘l.

compared with

SQ 18506

(S) in

2

Fig. 1. Comparison of results obtained by counting (b) 10 fields of cultures treated with SQ 18506.

(a) 30 fields and

Fig. 2. Effects of pentamidine (PA), pentostam (PE) and metronidazole (M) compared with nifurtimox (L).

concentrations it was no longer evident (Table III; see also Fig. 1). of other protozoicidal compounds Pentamidine, pentostam and metronidazole were compared with nifurtimox. Control cultures received only fresh medium 199 with serum or 0.1 y0 dimethyl sulphoxide. Metronidazole and pentamidine showed some effect on parasite replication at 100 pg ml-l; pentostam showed none. No compound was cytotoxic (Fig. 2). 6. Trials

t ; hi --iI :!t I: t I’I II 1 I 0 -Fig, 4. Effects of TETD

7. Trials

of MK

0 .Ol .l 1

0 1

T

S

(T) compared with SQ 18506 (S).

436 and tetraethylthiuram

disulphide

Two experiments were conducted using the nitroimidazole MK 436 and one using tetraethylthiuram disulphide (TETD), compared with SQ 18506. Control cultures received either 0.1% dimethyl sulphoxide or 0.1 “,/o ethanol. MK 436 had a pronounced trypanosomicidal effect, and was not significantly cytotoxic, at 10 pg ml-r (Fig. 3). TETD had a limited trypanosomicidal effect at 1 pg ml-i; at this dose it was cytotoxic (Fig. 4).

J. R. BAKER AND L. F. SELDEN Table II--Effect of various dionisii (stock P2) Compound

nitroheterocyclic

compounds

Concentration (bs ml-7

83

on BL cell cultures

infected

with

Percentage of cells infected*

Percentage of infected cells containing > 10 parasites*

Nifurtimox

1 1 1 10

23.0 i 4.3 62.3 3: 11.4 29.4 i 20.6

0 37.3 zt 17.0 14.1 f 9.5 0

MK 436 SQ 18506

10 O-01 o-1 ;:;

1.3 82.5 13-3 32.8 70.5

: 82.3 f 7.3 3.0 f 3.0 72.7 f0 14.7

Benznidazole

i: 0.7 f 12.5 i 2.4 if Il.0 9.3

1 1 :

0 0 21-l 1.4 1F rt 17.7 1.4

0 0 0

10 1 10

0 56.0 i 11.3 3.9 i 3.2

: 64.9 & 0

* Means f SEM (n = 3). Means of control cultures and > 74% respectively.

receiving

Table

compounds

III-Cytotoxic

Experiment number 1

2

effects

of nitroheterocyclic Drug

Control** SQ 18506

T.

only 0. l”/:, dimethyl

on BL cells infected

Concentration (cLg ml-9

Nifurtimox Benznidazole Control** SQ 18506

O-01 0.1 1 1 1 0.1

Nifurtimox

:

sulphoxide

7.6 all > 62%

with

T. dionisii

Number of cells in 10 microscope fields* 52.3 39.7 59.7 20.3 56.0 61.0 31.3 33.7 15.0 29.3

h f * rt f f * & i k

12.2 4.3 15.3 4.3 19.0 21.9 4.4 3.0 0.6 3.3

* Means & SEM (n = 3). ** Cultures receiving only drug solvent (O*10/0 dimethyl

sulphoxide).

Discussion In untreated cell cultures trypomastigotes of Trypanosoma dionisii infected a high proportion of BL cells in vitro, replicated as amastigotes, transformed into trypomastigotes after seven days, emerged from the cells and reinvaded other BL cells. Addition of suitable concentrations of nitroheterocyclic compounds known to be lethal to T. cruzi in tissue culture (GUTTERIDGE et al., 1978b) reduced to zero (or almost zero) the proportion of infected cells containing more than ten amastigotes (infection intensity) (see section 4 of the results). These proportions could be estimated fairly rapidly by examining ten microscope fields (magnification x 500) of a stained preparation, provided a reasonable number (> 30, preferably > 50) of cells was contained therein. Examining

more fields had no significant advantage and was, of course, slower. We could not eliminate considerable variation between experiments, and therefore always included cultures receiving (a) no additive other than fresh medium 199 and serum, (b) drug solvent(s) in equivalent concentration to that given to experimental cultures, and (c) a known trypanosomicidal drug at adequate concentration (e.g., nifurtimox at 10 pg ml-l or SQ 18506 at 1 pg ml-l). Our results with nifurtimox, benznidazole and SQ 18506 agreed fairly closely with those of GUTTERIDGE et al. (1978b), using T. cruzi in vitro. The different results we obtained in replicate experiments with nifurtimox at 1 /-Lg ml-l and SQ 18506 at 0.1 pg ml-l were probably due to these dosages being near the minimum effective levels. MK 436 at 10 pg ml--l (not tested by

a4

T. dionisii AS A DRUG TEST MODEL FOR T. cruzi

GUTTERIDGE et al., 1978b) completely eliminated infection and was not appreciably cytotoxic. MALANGA et al. (in press) have shown MK 436 to be trypanosomicidal in mice infected with T. cruzi, and it seems to warrant further investigation. We found that metronidazole (100 pg ml-l) slightly but definitely reduced infection intensity (but not rate), whereas GUTTERIDGE et al. (1978b) reported that its only effect was against extracellular trypanosomes. Metronidazole has only a transient effect on mice infected with T. cruzi (see PIZZI, 1961). Although SANTANA (1964) was cautiously ontimistic and PEREIRA (1965) found it “very in treating chronic human Chagas’s e&cient” disease. LEVI & AMATO NETO (1970) did not. and it is not generally accepted as an effective anti-T. cruzi agent (GUTTERIDGE, 1976; GUTTERIDGE et al., 1978b). Pentostam and pentamidine, used chemotherapeutically in leishmaniasis (STECK, 1972) but not known to be active against T. cruzi (see GUTTERIDGE, 1976), were virtually or entirely without effect in our system. TETD, an inhibitor of aldehyde dehydrogenase, interferes with threonine metabolism and is a potent inhibitor of growth of extracellular T. brucei, T. cruzi and T. dionisii in vitro at the maximum concentration used in this work (CROSS et al., 1975a, b and unpublished observations), but was ineffective in our tissue cultures. This indicates that our system evaluated the effect of compounds on intracellular parasites; also, when the test drug was added, most cellular invasion by parasites had already occurred. The lack of effect of TETD on intracellular parasites could be due either to its inability to reach them or to the use by these parasites of an alternate pathway in threonine metabolism. Our test system discriminated between compounds known to be active against T. cruzi and those which are not; amongst the former, levels of activity were similar to those reported against T. cruzi in tissue culture (GUTTERIDGE et aZ., 1978b). It might, therefore, serve as a preliminary “screening” test in vitro for possible activity against T. cruzi. Because of the variation inherent in the system, only compounds reducing the infection rate to zero (or to a mean value not exceeding zero by more than twice its standard error), and showing no cytotoxicity at the same dosage, should be selected for further investination. Differential counting of cells containing more than ten parasites gave no additional information and could be omitted. The graphic presentation of results used in Figs 3 to 6 was convenient for easy and rapid assessment. ‘3

Acknowledgements We are very grateful to Dr. J. A. Dvorak (National Institutes of Health) and Dr. R. A. Neal (Wellcome Research Laboratories) for supplying BESM cells ; to Dr. R. W. F. LePage (University of Cambridge) and our colleague Miss Nancy Lupton for BL cells; to Dr. Neal, Dr. W. E. Gutteridge (University of Kent), Dr. J. A. McFadzean and Dr. J. Hill (May and Baker Limited), Dr. Schiitz and Dr. Phimpe (Bayer AG), Dr. S. J. Lucania (Squibb Institute for Medical Research), Dr. I. Lenox-Smith (Roche Products Limited), Dr. M. H. Fisher (Merck,

Sharp and Dohme), Dr. A. Bryceson (Hospital for Tropical Diseases) and our colleague Dr. R. A. Klein for advice and help in obtaining drugs; and to Messrs. Bayer AG, May and Baker Limited, Merck and Company Inc., Roche Products Limited, E. R. Squibb and Sons Inc. and Wellcome Reagents Limited for generously supplying drugs. References Baker, J. R. (1976). Species of the subgenus Schizotrypanum other than Trypanosoma cruzi and their potential usefulness in the laboratory. Transactions of the Royal Society of TropicaZ Medicine and Hygiene, 70, 126-127. Baker, J. R. (1980). Primary isolate numbers of stocks of Trypanosoma (Schizotrypanum) species from Chiroptera in England. Systematic Parasitology, 1, 153-154. Baker, J. R., Green, S. M., Chaloner, L. A. & Gaborak, M. (1972). Trypanosoma (Schizotrypanum) dionisii of Pipistrellus pipistreZZus (Chiroptera) : intra- and extracellular development in vitro. Parasitology, 65, 251-263. Baker, J. R. & Liston, A. J. (1978). Trypanosoma (Schizotrypanum) dionisii: effect of various agents on attachment and entry to macrophages in vitro and on morphogenesis. Journal of General Microbiology, 104, 79-89. Bayles, A., Waitz, J. A. & Thompson, P. E. (1966). Growth of Trypanosoma cruzi in cultures of chick embryo cells, and effects of furazolidone and tris (p-aminophenyl) carbonium chloride. Journal of ProtozooZogy, 13 110-l 14. Bray, R. S. & Garnham, E. C. C. (1962). The Gremsa colophonium method for staining protozoa in tissue sections. Indian Journal of Malariology, 16, 153-155. Cross, G. A. M., Klein, R. A. & Baker, J. R. (1975a). Trypanosoma cruzi: growth, amino acid utilization and drug action in a defined medium. Annals of Tropical Medicine and Parasitology, 69, 513-514. Cross, G. A. M., Klein, R. A. & Linstead, D. J. (1975b). Utilization of amino acids by Trypanosoma brucei in culture: L-threonine as a precursor for acetate. Parasitology, 71, 311-326. Dvorak, J. A. & Hyde, T. P. (1973). Trypanosoma cruzi: interaction with vertebrate cells in vitro. I. Individual interactions at the cellular and subcellular levels. Experimenta Parasitology, 34, 268-283. Evans, D. A. (1978). Kinetoplastida. In: Methods of cultivating parasites in vitro. Taylor, A. E. R. & Baker, J. R. (Editors). London, New York, San Francisco: Academic Press, pp. 55-88. Fife, E. H. (1977). Trypanosoma (Schizotrypanum) cruzi. In: Parasitic Protozoa. Vol. 1, Kreier, J. P. (Editor). New York, San Francisco, London: Academic Press, pp. 135-173. Gaborak, M., Darling, J. L. & Gutteridge, W. E. (1977). Comparative drug sensitivities of culture forms of Trypanosoma cruzi and Trypanosoma dionisii. Nature, 266, 339-340. Gonnert, R., et seq. (1972). Nifurtimox: causal treatment of Chagas’ disease [and subsequent papers]. Arzneitmittel-Forschung, 22, (9a), 15631642.

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Accepted for publication 27th November, 1979.