Chronic Trypanosoma cruzi infection associated with low incidence of 1,2-dimethylhydrazine-induced colon cancer in rats

E.C. Oliveira1,3,6, M.S.B. Leite2, J.A.R. Miranda3, A.L.S.S. Andrade4, S.B. Garcia5, A.O. Luquetti3 and H. Moreira1

1 Departamento de Cirurgia,
2 Departamento de Patologia,
3 Laboratório de Pesquisa de Doencia de Chagas, Faculdade de Medicina and
4 Departamento de Saúde Coletiva, Instituto de Patologia Tropical e Saúde Pública, Universidade Federal de Goiás and
5 Departamento de Morfologia, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Brazil


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Experimental data have demonstrated that chronic infection with intracellular parasites may enhance resistance against some types of tumour. This phenomenon has not yet been demonstrated for experimental Trypanosoma cruzi chronic infection. This study investigated the effect of a specific colon cancer inducing drug, 1,2-dimethylhydrazine (DMH), on chronically T.cruzi infected Wistar rats. Infection was obtained by inoculation of 105 tripomastigote forms by subcutaneous (s.c.) route. Acute phase of the infection was monitored every other day by examination of a blood smear from each animal until negativation. In the early chronic phase of the infection, colon adenocarcinoma was induced by weekly s.c. injections of DMH at a dose of 20 mg/kg body weight for 12 weeks. 102 animals were divided in four test groups: 39 infected rats received DMH (group 1); 32 non-infected rats received DMH (group 2); 16 infected rats and 15 non-infected animals were used as control groups. Animals were killed 6 months after the first dose of DMH. The whole colon was removed and prepared for light microscopic examination. Twelve animals from group 1 and 22 from group 2 had colon adenocarcinomas, the proportion of cancer being 30.7 and 68.7%, respectively ({chi}2 = 10.16; P < 0.05). The relative risk of having a colon tumor in infected animals (group 1) was 0.45 (IC 95% 0.26–0.76), which is a protective risk compared with non-infected animals. These findings show that chronic infection with T.cruzi is associated with a lower incidence of DMH-induced colon cancer in rats.

Abbreviations: DMH, 1,2-dimethylhydrazine; PCNA, proliferating cell nuclear antigen; RR, relative risk; s.c., subcutaneous.


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
It is estimated that Chagas disease compromises 18 million people in Latin America. Many patients remain asymptomatic for life, but 10–20 years after infection around 7–15% develop digestive megaformations (megaesophagus and/or megacolon) (1,2). Gastrointestinal motility disturbances such as disphagia and long lasting constipation are the main complaints of patients. Constipation lasting <10 days in 70% of patients with chagasic megacolon is often observed (2,3). Motility alterations are due to destruction of neural cells of Meissner and Auerbach plexus during the acute phase of infection by the parasite Trypanosoma cruzi with an uneven distribution in the digestive tract (4).

Colorectal cancer is not a complication of acquired megacolon, as might be expected (57) considering the theory that constipation may prolong the contact of colonic cells with metabolic and dietary carcinogens thereby increasing the risk of cancer (811). Oliveira et al. (12) reported two cases of chagasic megacolon associated with colonic tumors. Both cases had tumors located in the transverse colon, i.e. proximally but not in the dilated portion (sigmoid colon and rectum). There are no reports of malignant tumors being found in the dilated segment of acquired megacolon. Interestingly, the most frequent dilated segment in chagasic megacolon is the rectosigmoid part (2,3) which is the same segment where most (two thirds) of the colorectal malignant tumors occur in the population as a whole (13). It is known that some protozoa, for example Toxoplasma gondii and Besnoitia jellisoni, enhance resistance against certain tumor cell types (14). Roskin and Exempliarskaia (15) were the first to report the influence of T.cruzi on tumor cell growth. Subsequent studies have been inconclusive with respect to the relationship between T.cruzi and cancer (1618). However, there are no data showing that experimental chronic infection by T.cruzi may influence the incidence specifically of colorectal tumors.

The clinical significance of this issue is that while chagasic megaesophagus is associated with a higher incidence of esophageal cancer of up to 9% (12), there is not one single reported case of chagasic megacolon with a malignant tumor located in the dilated segment of the colon (12). Chagasic megacolon causes severe constipation, and contrary to the theory that constipation prolongs the contact of possible carcinogens in the stool with the colonic mucosa (811), there have been no reports of a higher incidence of colon cancer in regions where the disease is endemic and where there is a high prevalence of megaformations, as is the case in central Brazil (2).

Much evidence suggests that various infectious agents may interfere in the natural history of neoplasia. Indeed, experimental data show that the host may be more susceptible or resistant to the appearance of tumors according to the stimulation of the immune system by the parasite (1923). This work reports the results of tumor incidence in animals challenged with T.cruzi infection before receiving an organ-specific carcinogen.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Animals
102 male Wistar rats bred in the Federal University of Goias' animal facilities, weighing 250 + 50 g and 90–120 days old were used. Water and a standard pellet diet (Labina, Ralston Purina do Brasil, São Paulo) were supplied ad libitum. All animals were kept in cages (five per cage) in a 12 h light/dark cycle.

Trypanosoma cruzi infection
Animals were infected with a T.cruzi strain isolated from a patient diagnosed to have chronic Chagas disease (24). Each animal received 105 tripomastigotes by subcutaneous (s.c.) route. Infection was monitored by checking parasitemia every other day until five consecutive examinations had failed to reveal trypanosomes. Parasitemia was assessed as described elsewhere (24). Chronic phase of the T.cruzi infection is defined by a very low level of parasites in peripheral blood, hardly detected by blood smear examination with light microscopy (25).

Carcinogen treatment
To induce colon neoplasm 1,2-dimethylhydrazine (DMH) (Aldrich, Milwaukee, USA) was employed. DMH was diluted in 0.001 M EDTA, pH 6.5 corrected with 1 N NaOH. Animals were injected with 20 mg/kg body weight weekly for 12 weeks by s.c. route.

Experimental design
Animals were divided into four test groups: group 1, 39 animals infected with T.cruzi + DMH; group 2, 32 non-infected animals injected with DMH; group 3, 16 animals infected with T.cruzi; group 4, 15 control animals. Groups 1 and 3 were infected with T.cruzi and after 100 days they were in the chronic phase of the infection with no detectable parasite in peripheral blood. Then groups 1 and 2, the latter of similar age, were injected with DMH. Six months after the first dose of DMH all animals were killed with ether overdose. The abdomen was opened and the colon was removed in totum, cleaned and then visible lesions were recorded. All macroscopic lesions as well as three longitudinal segments (15 mm) taken from proximal, middle and distal segments of each apparently normal colon were fixed in 10% (v/v) formalin, pH 7.0, cut into 4 µ thick sections and stained by hematoxylin/eosin for light microscopic examination.

This protocol was approved by the Animal Studies Ethical Committee of the Medical Faculty of the Federal University of Goiás.

Immunohistochemistry
To assess the proliferative activity of colonic epithelium and the distribution of proliferative cells in the crypt, proliferating cell nuclear antigen (PCNA) immunohistochemistry was performed according to the methods described previously (26). Two variables were scored, number of cells per crypt and PCNA-LI (PCNA labeling index), that express the percentage of PCNA-stained cells per crypt. We studied these parameters in both normal and transitional mucosa. Transitional mucosa is used to describe mucosa adjacent to colonic adenocarcinoma with characteristic morphological and histochemical features.

Statistical analysis
The number of animals to be included in each group was estimated considering one animal not challenged (group 2) to one animal challenged with T.cruzi infection (group 1) and expecting a tumor incidence of 90% in group 2 versus 50% in group 1. {chi}2 test was used to estimate differences in proportions of tumor incidence between groups. Relative risk (RR) of developing colon tumor (95% confidence interval) was calculated from the ratio of tumor incidence rate of group 1 (challenged by T.cruzi) to that of group 2 (not challenged). The number of cells per crypt and the PCNA-LI were compared by a two-sample t-test. Differences were considered statistically significant when P <= 0.05.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Trypanosoma cruzi infection
All 55 infected animals (groups 1 and 3) had detectable parasites in peripheral blood. Positivation occurred between 5 and 21 days post-infection and negativation after 21 and 53 days post-infection. A single peak was observed on the 13th day (mean 7090 parasites/ml) post-infection.

Neoplasia incidence
The proportion of cancer in groups 1 and 2 was 30.7 and 68.7%, respectively ({chi}2 = 10.16; P < 0.05) (Table IGo). Relative risk of colon cancer appearance in chronically infected animals with T.cruzi was 0.45 (CI 95% 0.26–0.76), which means a protector risk when compared with non-infected animals (group 2). Animals that received only DMH had a relative risk of 2.2 (1/0.45), i.e. a chance 120% higher of developing tumor than group 1, which was challenged by T.cruzi infection before exposure to DMH (Table IGo). In group 1, 12 out of 39 rats had malignant lesions (one with polyp, six with one flat lesion each, five with one stenosing tumor each); one rat had three flat benign lesions.


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Table I. Malignant tumor incidence in groups 1 (T.cruzi + DMH) and 2 (DMH)
 
On considering group 2, 22 rats had malignant tumors (four with one polyp each, four with one polyp plus one flat lesion each, 12 with flat lesions, one with stenosing tumor and one with stenosing lesion plus flat lesion) (Table IIGo); from those 12 rats with flat lesions three had syncronous tumors. Seven animals had benign flat tumors (four presented multiple lesions) and three had no macroscopic lesions. In all animals of group 3 no macroscopic lesions were observed. In group 4, five animals had one benign flat lesion each. Histopathology showed that all polyps and stenosing tumors were adenocarcinomas. Macroscopic flat lesions were of both types: benign (lymphoid aggregates) and malignant (adenocarcinoma). Seven animals in group 1 had nine flat lesions: six adenocarcinomas and three lymphoid aggregates. Twenty-four rats in group 2 had 46 flat lesions: 20 malignant and 26 benign.


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Table II. Types of macroscopic lesions found
 
All 20 malignant flat lesions in group 2 were associated with lymphoid plaques and four out of six in group 1 were associated with lymphoid tissue. Polyps were not associated with lymphoid aggregates. Macroscopic examination was not able to distinguish between benign and malignant flat lesions. Inflammatory infiltrate mainly by lymphocytes and plasma cells was observed in the muscularis propria of the colon in three, four and six animals of groups 1, 2 and 3, respectively. Inflammatory response was not associated with the presence of adenocarcinoma tumors in groups 1 and 2.

Three animals of group 1 had metastasis of the esophagus, stomach, liver, pancreas and testis. Metastasis were found in three rats of group 2 in the following organs: esophagus, lungs, small bowel and testis. Group 1 had five deaths but four occurred during the kill period. Group 2 had two deaths: one after the third dose of DMH and another during the kill period. All metastatic lesions were adenocarcinomas with the same histological pattern as primary colonic cancer.

Immunohistochemistry
Data showed that control and infected animals presented staining at the base of the colonic crypts. We observed a stepwise increase in the total number of cells per crypt and PCNA-LI in normal mucosa in group 2, but not in group 1. These features were also observed in the transitional mucosa in group 2 when compared with group 1 animals (Table IIIGo).


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Table III. Effects of DMH treatment on cells of crypt columns from distal colon in T.cruzi infected and non-infected animals
 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Trypanosoma cruzi chronically infected rats had a statistically significant lower level of drug-induced colon cancer than non-infected animals. Garcia et al. (27) reported a significant reduction in colon cancer in DMH-treated rats after chemical denervation of the distal colon by topical application of benzalkonium chloride. Destruction of neuron cells of the Meisner and Auerbach plexuses of the colon may be produced by T.cruzi infection (4) as well as by benzalkonium chloride (28). Hibbs et al. (14) reported similar results using chronically infected animals with intracellular protozoa (Toxoplasma gondii and Besnoitia jellisoni) and transplantable tumors. The higher frequency of flat lesions in group 2 may represent a stage between that of the normal cell and the pre-malignant lesion, considering that 30.7% (8/26) of benign lesions had slight to severe atypical glands. The latency period from first dose to kill may determine up to 100% of appearance of malignant tumors (29,30).

Both theories about colorectal carcinogenesis, adenoma-carcinoma sequence and de novo tumor, coexisted together in the same animal as well as reported by others (29,30). However, flat malignant lesions were more frequent than malignant polyps or stenosing lesions, supporting the theory that de novo cancer may be more common than is generally thought (2931). Chronic T.cruzi infection may have reduced the appearance of flat lesions in animals, i.e. group 3 (T.cruzi only) had no flat lesions while control animals (group 4) had five flat lesions, group 1 (T.cruzi + DMH) had nine lesions and group 2 (DMH) had 46 flat lesions. As all malignant flat lesions arose in lymphoid aggregates, and T.cruzi chronic infection is known to produce non-specific immunosuppression in man and experimental animals, it is possible that a link exists between them (32). Our data cannot confirm this theory but shows that the colonic mucosa in infected animals behaves differently to that of controls. Considering clinical reports, there are no reported cases of colonic tumors in acquired megacolon (12). Otherwise chagasic patients submitted to organ transplants present a higher incidence of lymphoid tumor than non-chagasic patients (33). Bocchi et al. (34) concluded that chronic infection, reactivation of the disease, immunosupression and the use of beznidazole (anti T.cruzi drug for human treatment) are responsible for the higher incidence of malignant neoplasia in heart transplanted Chagas patients. These may be important factors for lymphoproliferative disorders and Kaposi's sarcoma but not for all types of tumors. No one patient submitted to heart transplant had megaesophagus or megacolon that are considered contraindications for heart transplant in chagasic patients (33). Chagas disease affects millions of people in Latin America and there is no epidemiological evidence of a higher susceptibility to neoplasia.

Teixeira et al. (35) reported an incidence of 38% of lymphomas in T.cruzi infected rabbits after treatment with benznidazole but this finding was not reproduced and the drug is currently recommended as safe by Brazilian Health Ministry to treat infected children and acute cases (36).

Our results show that chronic T.cruzi infection is protective risk for colon adenocarcinoma. However, other types of tumors, such as cancer of the esophagus, should be tested to clarify the relationship between chronic T.cruzi infection and carcinogenesis. Trypanosoma cruzi strains may have tropism for different organs and elicit abnormal immunological responses in chronic patients which may up- or down-regulate the immune response against tumors (19,32).

The observation that animals in group 2 presented hyperproliferation with upward expansion of the proliferative compartment, that is a classically described characteristic feature of malignant progression, is in accordance with previous reports that the treatment of rats with DMH increases the colonic crypt length, labeling index and proliferative zone size (37). This was not observed in the animals of group 1. In the transitional mucosa surrounding the tumors, both groups of animals treated with DMH presented the crypts elongated and with higher proliferative activity in comparison with those in distant mucosa, which is also in accordance with other observations (38,39). Our data show that cell proliferation is increased in transitional mucosa in non-infected animals challenged with DMH (Table IIIGo). Probably chronic infection may enhance resistance in the digestive tract but not in other tissues. In support of this, no parasite was detected in histologic examinations of the colons of the T.cruzi infected animals. The presence of parasite seems not to be important for tumor development. However, microscopic detection of parasites at chronic tissue lesions may be less effective than PCR or immunohistochemistry (40). Nitric oxide appears to be increased in the acute phase of T.cruzi infection (41,42) and participates in neuron cell damage. Nitric oxide may favour tumor growth in the multifactorial carcinogenesis process (43), but a better understanding of the immunological mechanisms underlying chronic T.cruzi infection is needed.

Roskin and Exempliarskaia (15) were the first to report an anticancer effect of T.cruzi infection, which was followed by some controversial results (1618). Our results show a relative risk of appearance of colon tumor in group 3 (T.cruzi only) of 0.45, which is a protective risk. On the other hand, non-infected animals challenged with DMH had a 120% higher chance of tumors being present.

Our findings suggest that the colonic mucosa of T.cruzi infected animals is less altered by the action of DMH than that of uninfected animals. Whether this phenomenon is related to the lower incidence of tumors that we found and if it can be attributed to an improved immunity against neoplastic alteration in infected animals remains to be elucidated.


    Notes
 
6 To whom correspondence should be addressed Email: ecoliveira{at}cultura.com.br Back


    Acknowledgments
 
E.C.O. was supported by a grant from Conselho Nacional de Pesquisa (CNPq).


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received January 28, 2000; revised November 14, 2000; accepted November 20, 2000.





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