j. Comp. Path. 1990 Vol. i03

Malignant, Non-Hodgkin's Lymphomas in Trypanosoma cruzi-infected Rabbits Treated with

Nitroarenes

A. R . L. T e i x e i r a , R . S i l v a , E. Cunha Neto, J . M . Santana and L. V. R i z z o

Department of Pathology, Faculty of Health Sciences, University of Brasilia, 70 910 Bradlia, Brazil

Summary Use of 2-nitroimidazole, 5-nitrofuran and 5-nitroimidazole compounds in T. cruzi-infected rabbits resulted in a reduction in duration of parasitaemia in comparison with untreated, infected rabbits. The chronic myocarditis asso- ciated with Chagas' disease was not, however, prevented in nitroarene-treated rabbits; lymphocytic infiltrates associated with cardiac cell lysis, in the absence of parasites in situ, were present in both treated and untreated rabbits.

The carcinogenic effect of each trypanocidal nitroarene used in this study was also assessed. Administration of nitroarenes to rabbits resulted in the appearance of solid tumours in 37'8 per cent of animals that received drug therapy. Untreated, control rabbits in this series did not show tumour growth. Furthermore, malignant, mixed-cell type, non-Hodgkin's lymphomas were seen in 32'4 per cent of the treated rabbits. It seems that a direct relationship could be present between the presence of the nitro group, the trypanocidal cytotoxicity and the prevalence of tumours. Benznidazole cleared up parasit- aemias in the shortest time and was associated with 41.6 per cent oflymphoma growths, whereas MK-436 required twice as much time to clear blood paras!tes, and showed lymphomas in 25 per cent of experimental rabbits.

The demonstration of a high prevalence of malignant tumours in addition to the chronic myocarditis of Chagas' disease in nitroarene-treated rabbits is important since indiscriminate use of such compounds currently used to treat T. cruzi infections in man could increase the ris k of lymphoma.

Introduction Trypanosoma cruzi infection is an anthropozoonosis widely distributed in the Amer ican Continent . Over 150 species of mammals serve as natural and reservoir hosts for this flagellate protozoon (Foratini, 1980; Soulsby, 1987). These usually become infected by contamination with the faeces of an insect vector, the reduviid or tr iatomine bug. Twelve million people are estimated to be infected with T. cruzi, while 35 million are at risk of infection (PAHO report, 1970). Chagas ' disease is a chronic disease that may develop in some T. cruzi-infected hosts (Teixeira, 1979). It has been estimated that approxi- mate ly ha l f of the infected h u m a n population will develop Chagas' heart disease and/or "mega syndrome" affecting the oesophagus and colon (Prata,

0021-9975/90/05037 + 12 $03.00/0 © 1990 Academic Press Limited

38 A. R. L. Teixeira e t al.

1975). The morbidity and mortality that accompany chronic Chagas' disease inflict a severe economic burden on rural communities where the disease is endemic. Autoimmunity plays a role in the pathogenesis of Chagas' disease (Teixeira, 1986; Rizzo, Cunha Nero and Teixeira, 1989) making immuno- prophylaxis difficult to achieve.

Chemotherapeutic agents currently used to treat 7". cruzi infections are the nitroarene compounds which show marked trypanocidal activity (Grunberg, Beskid, Cleeland, Delorenzo, Titsworth, Scholes, Richle and Brener, 1968; Bock, Gonnert and Haberkorn, 1969). One of these, benznidazole (N-benzyl- 2-nitro-imidazoleacetamide) has been used to treat Chagas' disease in Brazil (Can~ado and Brener, 1979), while another, nifurtimox {4-(5-nitro-phurylide- neamino)-tetrahydro-4-4-1, 4-thiazine-l-l-dioxide}; has been used to treat 7-. cruzi-infected individuals in other Latin-American countries (Cerisola, 1969). In addition, the nitroarene MK-436 {3-(1-methyl-5-nitroimidazol-2- yl)-3d, 4, 5, 6, 7d-hexahydro-1, 1-benzisoxazoline}, which has shown trypano- cidal activity in vitro, has been used as a therapeutic agent in experimental Chagas' disease.

In view of the severe cytotoxicity that has been described for several nitroarenes (Headley, Klopp, Michie, Erturk and Bryan, 1981; Teixeira, Jabur, Souto Maior and Sol6rzano, 1983b; Ferreira and Ferreira, 1986), we decided to assess the effects of the administration to rabbits of three of these compounds known to have trypanocidal activity.

M a t e r i a l s a n d M e t h o d s

Parasite

The Ernestina stock of T. cruzi used in this study was isolated from a child with acute Chagas' disease in S~o Felipe, Bahia, Brazil, in 1964. The parasite has been maintained in our laboratory by serial passage in tissue culture (Teixeira, Figueiredo, Rezende Filho and Macedo, 1983a). Free-swimming trypomastigotes collected by centrifugation (1000 Xg for 10 min) of the nutrient medium were used for the experimental infections.

Inoculation of Rabbits with Trypomastigotes ofT. cruzi and Detection of Parasitaemias

Twenty-seven, 2-month-old outbred New Zealand white male and female rabbits were inoculated subcutaneously with 1 x 106 trypomastigotes per kg body weight. Parasitaemias in 7-. cruzi-infected rabbits were monitored by xenodiagnosis, which consisted of 20 first instar nymphs of the reduviid bug, Dipetalogaster maximus, obtaining blood meals from each of the infected rabbits. This was repeated every 15 days for 6 months and every month thereafter. A positive diagnostic test was indicated by the presence of the parasite in the faeces of D. maximus fed the rabbit's blood. Faeces were examined 30 and 60 days after exposure.

Administration of Nitroarenes in Rabbits

In these experiments, each drug was given by intraperitoneal (i.p.) injection at the dose used to treat Chagas' disease in man (Bock et al., 1969; Can~ado, 1973). MK-436 (80 mg per kg per day for 60 days) was administered to six T. eruzi-infeeted rabbits (group A) and six non-infected controls (group B) (Table 2). Nifurtimox (8 mg per kg per day for 60 days) was administered to 7". cruzi-infected rabbits (group D) and a further six non-infected control animals (grOup E) (Table 3). Benznidazole (8 mg per

Nitroarenes in T. cruzi infections 39

kg per day for 60 days) was administered to seven T. cruzi-infected rabbits (group G) and six non-infected controls (group H) (Table 4). Nifurtimox (Bayer Pharmaceutical Industries, Germany) and MK-436 (Merck Sharp and Dohme Laboratories, Switzerland) were gifts from Prof. Aluizio Prata, Nucleo de Medicina Tropical, Universidade de Brasilia. The chemically pure benznidazole used in this study was a gift from Dr Ivo Ferreira, Director, Roche Pharmaceutical Industries, Rio deJaneiro. Saline suspensions of each drug were used since these compounds are insoluble in dimethylsulphoxide and other organic solvents.

The remaining eight untreated, T. cruzi-infected rabbits were kept as positive controls (Table 1). Additional control animals (groups C, F and I) consisted of 18 non-infected, untreated rabbits (Tables 2, 3 and 4). All rabbits were fed commercial pellets and water ad libitum and kept in individual cages throughout the investigation.

Post-mortem Studies Complete necropsies were performed on each experimental rabbit. All organs and tissues were examined for gross lesions and case material was fixed in 10 per cent buffered formalin. Representative sections, 5 gm thick, were taken from tissues, embedded in paraffin wax and stained with haematoxylin and eosin (HE).

Statistical Analysis Student's t-test was used for group analysis.

R e s u l t s

Trypanocidal Activity of Nitroarenes

The administration of nitroarenes to rabbits infected with 7". cruzi resulted in a curtailment of parasitaemias demonstrable by xenodiagnosis, as shown in Fig. 1. On average, parasitaemias in rabbits with T. cruzi infections lasted 5 months, after which time, parasites disappeared spontaneously. The admin- istration of MK-436 to T. cruzi-infected rabbits reduced duration of parasit- aemia by approximately 50 per cent to 3 months. Nifurtimox and benznidazole reduced the duration of parasitaemia by 62"5 and 75 per cent, respectively, in comparison with untreated T. cruzi-infected animals.

Chagas' Myocarditis and Myositis

The Chronic myocarditis of Chagas' disease was observed in seven untreated T. cruzi-infected rabbits (Table 1). The cardiac inflammatory lesions in these animals were indistinguishable from those described in the heart of every rabbit chronically infected with T. cruzi that received nitroarene therapy (Tables 2, 3 and 4). Lymphocytic infiltrates associated with cardiac cell lysis in the absence of the parasite in situ were observed in every T. cruzi-infected animal in this study (Fig. 2). The destructive inflammatory lesions and fibrous replacement were focal ( + ), patchy ( + +) or diffuse (+ + + ). In addition, discrete lymphocytic infiltrates were present in sections taken from pectoral, psoas and gluteus striated muscle and from smooth muscle layers of the digestive tract. None of these lesions were seen in non-infected rabbits that received nitroarene therapy or in the untreated, uninfected control animals.

40

Fig. 1.

A. R. L. T e i x e l r a et al.

i l '(" " I i

liii~ ! .i#i~.!i!.!iJ.~. !~i.~i~ !~i. #.i~;!i~!i! !~.. ii!~ ~.~.~iiL. ~.!!~.~ ~i. I

l®i

t I I

I I I I I I I I 2 3 4 5 ? 8

Months

Parasitaemias in rabbits with Trypanosoma cruzi infections. (a) Untreated rabbits (n=8) that underwent spontaneous recovery by 5 months post-infection; (b) MK-436-treated rabbits (n= 6); (c) nifurtimox-treated rabbits (n = 6); and (d) henznidazole-treated rabbits (n= 7). Stippled lanes indicate a phase of infection with patent parasitaemias; lines show prepatent and subpatent phases of infection; Bars indicate standard deviation of the means and arrows show beginning of nitroarene therapy.

Tumour Growth

Of 12 rabbits that received 80 mg per kg of MK-436 by i.p. injection for 60 days (Table 2), three (25 per cent) developed tumours in the spleen, lymph nodes, liver, kidneys, lungs and in lymphoid tissue along the intestinal tract. Fig. 3 is a section taken from a lymph node of rabbit 12 that shows invasive neoplastic growth replacing normal tissue. Large and small lymphocytes with clumped chromatin, may be seen. Mitotic figures are frequent. Large clear cells with "cart wheel" nuclei are present, some of which show recognizable nucleoli. In addition, one of the uninfected rabbits that received MK-436 developed an adenocarcinoma of the s tomach with metastases in the lung and liver.

The results of nifurtimox administration are shown in Table 3. Four rabbits (33 per cent) showed lymphomas involving spleen, lymph nodes, liver, lungs and other lymphoid tissue in the body. Fig. 4 is a section of' the kidney from the nifurtimox-treated rabbit number 33 and shows cortical tissue replaced by invasive mixed-cell populations of a malignant, non-Hodgkin 's lymphoma.

Table 4 illustrates the effect ofbenznidazole administration. Five rabbits (41 per cent) showed tumours invading lymphoid structures in the body. Fig. 5 is a section of spleen from benznidazole-treated rabbit number 46, which shows aberrant, mixed, neoplastic lymphoid cell populations replacing normal tissue. Rabbit 57, treated with benznidazole, had an adenocarcinoma of the colon at post-mortem examination.

N i t r o a r e n e s i n 7". e r u z i i n f e c t i o n s

Table 1 M y o c a r d i t i s and s u r v i v a l a f t e r Trypanosorna cruzi inocu la t ion of rabbi ts

41

Rabbit Sex T. cruzi* Histopathologieal findings Survivalt number inoculum (days after treatment

Myocarditis Neoplasia mean 4- s.d. )

1 F 2,4 + + + - - 275 2 M 2'~ - - - - 540 3 M 2.0 + + - - 730 4 M 2 '3 + - - 1785 5 F 1.9 + + - - 167 6 F 2"0 + - - 1365 7 M 2-5 + + - - 1095 8 F 2-3 + + + - - 150

= 765.4- 639

* = T w o - m o n t h - o l d New Z e a l a n d white r abb i t s were inocula ted subcutaneous ly wi th 1 × 10 ~ tissue culture der ived t rypomast igo tes per kg body weight ; t = D a y zero (0) was indicated by last d a y of n i t roarene t h e r a p y admin is te red to 7-, cruzi-infected rabbi t s shown in Tables 2, 3 and 4,

Table 2 Ef fect o f MK-436 a d m i n i s t r a t i o n to r a b b i t s in fec ted wi th Trypanosoma cruzi and to n o r m a l

rabbi t s

Rabbit Sex T. eruzi* Histopathologieal findings Suroival~ (days number inoeulum after treatment;

Myocarditis** Neoplasia mean :t: s.d.)

Group A: Treated, T. cruzi-infected rabbits 9 F 1 "7 + + - - 1248

10 M 1'9 + + - - 690 11 F 2'1 + + - - 960 12 M 1.6 + L y m p h o m a 392 13 F 1'8 + + + - - 450 14 M 2 '0 + + - - 720

~ = 743-4-320

Group B: Treated, non-infected rabbits 15 M None - - L y m p h o m a 460 16 F None - - - - 1430 17 M None - - - - 992 l 8 M None - - L y m p h o m a 330 19 F None - - - - 870 20 F None - - Adenoca rc inoma

of s tomach 810 ~ , = 815-4-393

Group C: Untreated, control rabbits 21 to 26 None - - - - ~ 1 3 5 0 + 4 2 0

* = T w o - m o n t b - o l d New Z e a l a n d white r abb i t s were inoculated wi th I x 10 ~ t rypomast igotes of T. cruzi per k g body weight . Each r a b b i t in g roups A a n d B received da i ly in t raper i tonea l injections of 80 mg per kg of M K - 4 3 6 for 60 days . ** + = focal l ymphocy t i c infiltrates and h e a r t cell lysis; + + = pa tchy lymphocyt ic infi l t rates, ta rge t cell lysis a n d fibrosis; + + + = diffuse lymphocy t i c infiltrates, h e a r t cell lysls and fibrosis. t = D a y zero (0) for every exper imenta l a n d control g roup was indicated by last d a y ofn i t roarene therapy. C o m p a r i s o n of ave rage survivals: g r o u p A vs B, P>0'50; g r o u p A vs C, P < 0 , 0 2 ; g roup B vs C, P < 0 ' 0 5 .

4 2 A . R . L . T e i x e i r a et al.

Table 3 Effect of n i f u r t i m o x a d m i n i s t r a t i o n to Trypanosorna eruzi-infected r a b b i t s a n d to n o r m a l

r a b b i t s

Rabbit Sex T. cruzi* Histopathological findings Survivalt (days number inoculum after treatment;

Myocardltis** Neoplasia mean 4- s.d.)

Group D: Treated, T. cruzi-infected rabbits 27 M 2'0 + + L y m p h o m a 665 28 M 1.8 + - - 1095 29 F 1.7 + + + - - 1345 30 F 1.9 + + L y m p h o m a 277 31 M 2.1 + + - - 410 32 F 1.8 + + + - - 370

R = 693 4- 434

Group E: Treated, non-infected rabbits 33 F None - - L y m p h o m a 450 34 F None - - - - 1470 35 M None - - L y m p h o m a 460 36 M None - - - - 665 37 F None - - - - 9 l 2 38 M None - - - - 382

N2= 7234-4.14

Group F: Untreated, control rabbits 39 to 44 None - - - - X = 1385 :t: 392

*Two-month -o ld New Zealand white rabbi ts were inoculated with 1 x 10 ~ t rypomast lgotes of 7". oruzi per kg body weight. Each rabbi t in groups D and E received dai ly in t raper i toneal injections of 8 mg p e r kg or a saline suspension of nifurtimox for 60 days. ** + = focal lymphocyt ic infil trates and hear t cel l lysis; + + --- pa tchy lymphocyt ic infiltrates, target cell lysis and fibrosis; + + + = diffuse lymphocy t i c infi l t rates, hear t cell lysis and fibrosis. I" = Day zero (0) for every exper imenta l and control group was i nd i ca t ed by last day of ni t roarene therapy. Compar ison of average survivals: group D vs E, P > 0"50; g roup D vs F, P < 0'01; g roup E vs F, P<0"01 .

The 18 normal, non-infected, untreated control rabbits showed no tumour growth at post-mortem. Furthermore, blood smears taken periodically from every control and experimental rabbit showed no neoplastic cells.

There was no significant difference in the survival of 7". cruzi-infected or non-infected rabbits that had received nitroarene treatment. However, both groups of rabbits receiving nitroarene therapy survived for a shorter period than normal, untreated control animals; these differences in survival being statistically significant (Tables 2, 3 and 4).

D i s c u s s i o n

This paper reports on malignant, non-Hodgkin's lymphoma occurr ing in rabbits after administration of nitroarenes. It shows further that therapeutic administration of nitroarenes in T. cruzi-infected rabbits did not arrest the destructive myocarditis of Chagas' disease, even though these drugs considera- bly reduced the duration of parasitaemias.

N i t r o a r e n e s i n T. c r u z i i n f e c t i o n s 4B

Table 4 Effect o£ b e n z n l d a z o l e a d m i n i s t r a t i o n to Trypanosoma cruzi-infected rabbits and to n o r m a l

rabbi t s

Rabbit Sex T. eruzi* Histopathological findings Survival t (days number after treatment;

Myocarditis* * Neoplasia mean 4- s.d. )

Group G." Treated, T. cruzi-infected rabbits 45 F 2'0 + + + - - 150 46 F 2' I + + + Lymphoma 49 47 F 2'2 + + - - 1850 48 M 2'0 + Lymphoma 160 49 F 1'9 + + + - - 175 50 M 1.8 + + - - 1220 51 M 2'4 + + Lymphoma 72

~ = 5 5 2 ± 7 1 4

Group H: Treated, non-infected rabbits 52 F None - - - - 1275 53 M None - - - - 670 54 F None - - - - 850 55 M None - - Lymphoma 650 56 F None - - Lymphoma 905 57 M None - - Adenoeareinoma

of colon 1625 ~ = 878±457

Group I: Untreated, control rabbits 58 to 63 None - - - - ~ = 1496+353

* = Two-month-o ld New Zea land white rabbi ts were inoculated with 1 x 106 trypomastigotes of 7". eruzi per kg body weight; Each rabb i t in groups G and H received dai ly intraperi toneal injections of 8 mg per kg of a sal ine suspension of benznidazole for 60 days. ** + = focal lymphocytic infiltrates and heart cell lysis; + + = pa tchy lymphocyt ic infiltrates, target cell lysis and fibrosis; + + + = diffuse lymphocytic infiltrates, hear t cell lysls and fibrosis, t = Day zero (0) for every experimental and control group was indicated by last day o fn i t roa rene therapy. Compar ison & a v e r a g e survivals: group G vs H, P > 0 ' 2 ; group G vs I, P < 0 ' 0 5 ; g roup H vs I, P < 0 ' 0 5 .

relationship between undetectable parasitaemias and the cure of T. cruzi infections in rabbits treated with trypanocidal nitroarenes. An answer to this question might come from results in T. cruzi-infected animals (Fig. 1) in which infections cleared spontaneously within 6 months. These 7-. cruzi-infected rabbits were indistinguishable from those that received nitroarene therapy in that they showed negative parasitaemias. With respect to the immune res- ponses against the parasite antigens, treated and untreated animals were also indistinguishable. Previous work has shown that humoral antibody responses to the parasite antigens persisted throughout in untreated as well as in nitroarene-treated 7". cruzi-infected rabbits (Teixeira et al. 1983b; Teixeira, C6rdoba, Souto Major and Sol6rzano, 1989). Although delayed-typ~immune responses to specific antigens may be temporarily suppressed during admini- stration of either nifurtimox (Lelchuck, Cardoni and Fuks, 1977) or benznida- zole (Teixeira et al. 1983b) in T. cruzi-infected animals, those cell-mediated immune reactions could continue after discontinuation of drug-therapy.

44 A . R . L . T e i x e i r a e t al .

Fig. 2.

Fig. S.

Chronic myoearditis of Chagas' disease in rabbit 32 that received nifurtimox therapy: Lymphocytic infihratcs are associated with myoeyte lysis in absence of T. cruzi. HE × 160.

Lymph node fi'om a MK-436-treated, 7-. cruzi-infected rabbit shows (L) large and (S) small lymphocytes with clumped chromatln. Mitotic figures are frequent. Large dear cells with "cart wheel" chromatin are present, some of which show recognizable nucleoli. HE × 400.

N i t r o a r e n e s in T. c r u z i i n f e c t i o n s 4-5

Fig. 4.

Fig. 5.

Kidney from a nifurtimox-treated rabbit showing cortical tissue replaced by invasive, mixed-cell populations from a malignant, non-Hodgkin's lymphoma. HE x 400.

Spleen from a bcnznidazolc-treated rabbit showing aberrant, neoplastic, mlxed-lymphoid cell populations replacing normal tissue, HE x 160.

46 A . R . L . Teixeira e t al.

In general, the observations in the animal model parallel results in series of patients naturally infected and treated either with benznidazole (Grunberg et al., 1968; Cangado and Brener, 1979) or with nifurtimox (Book et al., 1969; Cerisola, 1969). However, individuals that had shown parasitaemia before nitroarene administration may yield positive xenodiagnosis subsequent to treatment. Furthermore, chronic T. cruzi infections in man may show recrudes- cence of parasitaemia after administration of immunosuppressive agents (Almeida, Cunha, Chapadeiro and Lopes, 1972; Kohl, Picketing, Frankel and Yaeger, 1982). In view of the evidence available we believe that persistence of immune responses in nitroarene treated 7-. cruzi-infected individuals indicate subpatent, active ongoing infections. These persisting subpatent infections might play a role in the outcome of Chagas' heart disease, which could be exacerbated by antigenic stimulation of autoimmune mechanisms involved in its pathogenesis (Teixeira, 1979; Rizzo et al., 1989).

Of considerable interest is the demonstration of drug-induced malignant lymphomas in 25 per cent of the rabbits that received the nitroarene MK-436, in 33 per cent of those that received the nifurtimox and in 41"6 per cent of those rabbits that received benznidazole. A direct relationship could be drawn between the nitroderivative trypanocidal or cytotoxic activity and incidence of tumour growths in treated rabbits. Benznidazole, which curtailed parasitae- mias in the shortest time, was also associated with the highest incidence of lymphomas.

In recent decades, several nitroarenes have been shown to possess a variable degree of carcinogenic activity (Erturk, Price, Morris, Cohen, Leith, Esch and Crovetti, 1967; Cohen, Erturk, Crovetti and Bryan, 1973). However, some nitroarenes, such as the 1-{(5-nitrofurfurylidene)-amino)-hydantoin} (Morris, Price, Lalich and Stein, 1969) and the {4-(5-nitro-phurylideneamino)-tetra- hydro-4-4-1,4-thyazine-l-l-dioxide} (nifurtimox) have not been shown to possess carcinogenic activity (Steinhoff and Grundmann, 1972). The cytotox- icity and genotoxicity associated with nitroarenes seem to depend on the binding of electrophilic radicals to macromolecules (Knox, Knight and Edwards, 1981). Also, it has been shown that trypanocidal nitroarenes undergo enzymatic reduction of the nitro-group and generate free electrophilic radicals such as nitro-anions, hydroxyl, singlet oxygen and hydrogen peroxide (Docampo, Mason, Mottley and Muniz, 1971; Moreno, Docampo, Mason and Leon, 1982). Nevertheless, information in relation to their possible carcino- genic activity is scarce in the literature.

in the experiments reported here, over 32 per cent of the rabbits that were given nitroarene therapy developed a single type of malignant, widely invasive, mixed-cell type, non-Hodgkin's lymphoma. In addition, 5"4 per cent of those animals that had received drug therapy showed adenocarcinomas of the digestive tract with metastases elsewhere. The spontaneous incidence of solid tumours in the rabbit species is given as 2"7 per cent in one report (Weisbroth, Flatt and Kraus, 1974). In this series, the incidence of malignant, non- Hodgkin's lymphomas in 12 out of 37 rabbits (32 per cent) that received nitroarene therapy compared with none of the 18 untreated control rabbits, far exceeds the expected incidence of spontaneous tumours.

Nitroarenes in T. c r u z i infections 47

Acknowledgments

This work received financial support from Conselho Nacional de Desenvolvimento Cientffico e Tecnol6gico do Brasil, CNPq. We are indebted to Martha Zappal~, Edwin Sol6rzano, Jos6 Carlos C6rdoba, Eliane Jabur, Auxiliadora Calixto and Inks Souto Major, who worked in this laboratory during the five year period in which these studies were carried out. The technical aid ofJos~ Tavares, Jemima Santos and Elizabete Lima is also deeply appreciated. We are grateful to Mr Evandro M. Gama who typed the manuscript.

References

Almeida, H. O., Cunha, J. G., Chapadeiro, E. and Lopes, E. R. (1972). Parasitismo do esofago e cora~o do chag~isico cr6nico portador de doen~a de Hodgkin em uso de immunossupressores. Revista Sociedade Brasileira Medicina Tropical, 6, 367-375.

Bock, M., Gonnert, R. and Haberkorn, A. (1969). Study with Bay 2502 on animals. Boletin Chileno Parasitologia, 24, 13-19.

Can~ado, J. R. (1973). Aspectos clinicos da padroniza~-o dos efeitos terap&uticos da Doen~a de Chagas. Revista Goiana de Medicina, 9, 217-232.

Canfado, J. R. and Brener, Z. (1979). Terap~utica. In: Trypanosoma cruzi e Doenfa de Chagas. Z. Brener and Z. Andrade, Eds. Guanabara Koogan, Rio de Janeiro, 362-424.

Cerisola, J. A. (1969). Serologic findings in patients with acute Chagas' disease treated with Bay 2502. Boletin Chileno Parasitologia, 24, 54-59.

Cohen, S. M., Erturk, E., Crovetti, A.J. and Bryan, G. T. (1973). Carcinogenicity of 5-nitrofurans, 5-nitroimidazoles, 4-nitrobenzenes and related compounds. Journal of the National Cancer Institute, 51,403-417.

Docampo, R., Mason, R. P., Mottley, C. and Muniz, R. P. A. (1971). Generation of free radicals induced by Nifurtimox in mammalian tissues. Journal of Biological Chemistry, 256, 1093-1098.

Erturk, E., Price, J .M., Morris, J.E., Cohen, S., Leith, R.S., Esch, A.M. and Crovetti, A.J. (1967). The production of carcinoma of the urinary bladder in rats by feeding N-{4-(5-nitro-2-furyl)-2-thiazolyl} formamide. Cancer Research, 29, 1998-2002.

Ferreira, R. C. C. and Ferreira, L. C.S. (1986). Mutagenicity of Nifurtimox and Benznidazole in the salmonella-microsome assay. Brazilian Journal of Medical and Biological Research, 19, 19-25.

Foratini, O.P. (1980). Biogeografia, origem e distribuig~o da domicilia~o de triatomlneos no Brasil. Revista de Sa(tde P~blica, Sgo Paulo, 14, 265-299.

Grunberg, E., Beskid, G., Cleeland, R., Delorenzo, W. F., Titsworth, E., Scholes, H.J., Richle, R. and Brener, Z. (1968). Ar~tiprotozoon and antibacterial activity of 2-nitroimidazote derivatives. Antimicrobial Agents and Chemotherapy, 7, 513-519.

Headley, D.B., Klopp, R.G., Michie, P.M., Erturk, E. and Bryan, G.T. (1981). Temporal comparison of immune status and target organ histology in mice fed carcinogenic 5-nitrofurans and their nor-nitro analogs. Cancer Research, 41, 1397-1401.

Knox, R.J., Knight, R. C. and Edwards, D. I. (1981). Interaction of nitroimidazole drugs with DNA in vitro: structure-activity relationships. Brit#h Journal of Cancer, 44, 741-745.

Kohl, S., Pickering, L. K., Frankel, L. S. and Yaeger, R.G. (1982). Reactivation of Chagas' disease during therapy of acute lymphocytic leukemia. Cancer, 50, 827-828.

Lelchuck, R., Cardoni, R.L. and Fuks, A.S. (1977). Cell-mediated immunity in Chagas' disease: Alterations induced by treatment with a trypanoeidal drug (Nifurtimox). Clinical and Experimental Immunolo~, 3, 43~ ~38.

48 A.R.L. Teixeira e t al.

Moreno, S.J.N., Docampo, R., Mason, R. P. and Leon, W. (1982). Different behavior of Benznidazole as free radical generator with mammalian and Trypanosoma cruzi microsomal preparations. Archives of Biochemistry and Biophysics, 218, 585-591.

Morris, J.E., Price, J.M., Lalich, J.J. and Stein, R.J. (1969). The carcinogenic activity of some 5-nitrofuran derivatives in the rat. Cancer Research, 29, 2145-2156.

Pan American Health Organization. (1970). Report of a study group on Chagas' disease. Pan American Health Organization Scientific Publication, 195.

Prata, A. (1975). Natural history of chagasic cardiomyopathy. Pan American Health Organization Scientific Publication 301, 191-193.

Rizzo, L.V., Cunha Nero, E., and Teixeira, A.R.L. (1989). Autoimmunity in Chagas' disease: Specific inhibition of reactivity ofCD4 + T cells against myosin in mice chronically infected with Trypanosoma cruzi. Infection and Immunity, 57, 2640-2644.

Soulsby, E.J.L., Ed. (1987). Immune Responses in parasitic infections: hnmunology, immunopathology and immunoprophylaxis. CRC Press, Boca Raton, FL, pp. 25-118.

Steinhoff, D. and Grundmann, E. (1972). Test of carcinogenicity of nifurtimox on oral and subcutaneous administration to rats. Arzneim.-Forsch (Drug Research), 22, 1607-1612.

Teixeira, A. R. L. (1979). Chagas' disease: Trends in immunological research and prospects for immunoprophylaxis. Bulletin of the World Health Organization, 57, 697-710.

Teixeira, A. R. L. (1986). Animal model of human disease: Chagas' disease in isogenic III/J rabbits. American Journal of Pathology, 124, 363-365.

Teixeira, A. R. L., Figueiredo, F., Rezende Filho, J. and Macedo, V. (1983a). Chagas' disease: A clinical, parasitological, immunological and pathological study in rabbits. American Journal of Tropical Medicine and Hygiene, 32, 258-272.

Teixeira, A. R. L.,Jabur, E., Souto Major, I. C. and Sol6rzano, E. (1983b). Altera~o da resposta imune mediada por cfilulas durante o tratamento em Benznidazole. Riv#ta da Sociedade Brasileira de Medieina Tropical, 16, 11-22.

Teixeira, A. R. L., C6rdoba, J. C., Souto Major, I. and Sol6rzano, E. (1989). Chagas' disease: Lymphoma growth in rabbits treated with Benznidazole. American Journal of Tropical Medicine and Hygiene, (in press).

Weisbroth, S.H., Flatt, R.E. and. Kraus, A.L., Eds. (1974). The Biology of the Laboratory Rabbit. Academic Press, New York, pp. 333.

[Received, July 14th, 1989] [Accepted, November 2nd, 1989]