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Trypanosoma cruzi: The effects of dehydroepiandrosterone (DHEA) treatment during experimental infection
Acta Tropica 95 (2005) 109–115
Trypanosoma cruzi: The effects of dehydroepiandrosterone (DHEA) treatment during experimental infection Carla Domingues dos Santos ∗ , M´ıriam Paula Alonso Toldo, Jos´e Cl´ovis do Prado J´unior Laborat´orio de Parasitologia, Departamento de An´alises Cl´ınicas, Toxicol´ogicas e Bromatol´ogicas, Faculdade de Ciˆencias Farmacˆeuticas de Ribeir˜ao Preto FCFRP-USP, Universidade de S˜ao Paulo, Avenida do Caf´e s/no, 14040-903 Ribeir˜ao Preto, SP, Brazil Received 1 April 2005; received in revised form 7 April 2005; accepted 12 May 2005 Available online 13 June 2005
Abstract The aim of this study was to evaluate the efficacy of the immunomodulator dehydroepiandrosterone (DHEA) in the treatment of Trypanosoma cruzi infection and the possible biochemistry alterations in male and female Wistar rats. DHEA also known as the steroid of multiple actions has attracted distinct medical areas. Prior studies show that DHEA enhances immune responses against a wide range of viral, bacterial and parasitic pathogens. Furthermore, administration of DHEA seems to protect animals against obesity and diabetes. Male animals subcutaneous treated with 40 mg/kg body weight/day of DHEA displayed a significant reduction in blood parasites during parasitaemia peak, when compared to untreated animals (P < 0.001). For female group parasitaemia was also reduced although values are not statistically significant (P > 0.05). Sexual dimorphism was also observed, since females displayed lesser parasitaemia levels compared to males group treated (P > 0.05) and untreated (P < 0.001). Enhanced leucocytes number was observed in control females when compared to control males (P < 0.05). DHEA treatment did not triggered any significant alterations in leucocytes levels (P > 0.05). DHEA administration induced an enhanced number of macrophages in infected male (P < 0.01). DHEA administration causes a decrease in glucose (P < 0.001). Cholesterol and tryglicerides levels did not display results statistically significant (P > 0.05) during the treatment. These results suggest that DHEA treatment enhances the immune response as evidenced here by reduced levels of parasites. Up-regulation of the immune system by exogenous DHEA may be useful in the treatment of American tripanosomiasis. © 2005 Elsevier B.V. All rights reserved. Keywords: Dehydroepiandrosterone; Trypanosoma cruzi; Glucose; Peritoneal macrophages; Parasitemia; Leucocytes
1. Introduction ∗
Corresponding author. Tel.: +55 16 602 4153; fax: +55 16 602 4163. E-mail address: [email protected] (C.D. Santos). 0001-706X/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.actatropica.2005.05.005
Chagas’ disease is endemic in Latin America, affecting 16–18 million people, with more than 100 million exposed to the risk of infection (WHO, 1991).
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American trypanosomiasis is a protozoan infection caused by Trypanosoma cruzi and is one of the most important public health problems in Latin America. Gender and the corresponding sex steroids have shown several effects on immune response in a wide range of different species including humans, rodents and birds (Schuurs and Verheul, 1990). A bulk of evidence shows that sexual dimorphism plays an important role in the modulation of the immune response when hosts are exposed to distinct etiological agents. This means that a bi-directional relationship between endocrine and the immune system exists, in order to maintain homeostasis (Syrt and Sugarman, 1991; Prado et al., 1998, 1999). Women are more efficient in clearing peripheral parasitaemia these facts are probably linked to sexual dimorphism and steroid hormones (Brabin and Brabin, 1992). Estrogens exert a positive action on B cell proliferation, leading to enhanced immunoglobulin production. For this reason, women evade the pathological actions of a wide range of etiological agents more rapidly. Some of them produce severe illnesses in women such as Toxoplasma gondii and Trichomonas vaginalis (Roberts et al., 2001). DHEA is one of the steroid hormones produced by the adrenal cortex. DHEA sulphatase (DHEA-S) plays an important role in converting most all DHEA in DHEA sulphate (DHEAS), the latter form being one of the most common substances found in the circulation (Parker, 1995). Just before birth and again at puberty, DHEA starts to be produced in elevated concentrations, reaching its peak at around 20–30 years of age, followed by a progressive decline (Baulieu, 1996). DHEAS is also implicated in age-related changes in the immune system (Loria et al., 1988) and has been associated with disease susceptibility (Shealy, 1995). Currently, DHEA is one of numerous immunomodulators undergoing clinical evaluation as a potential treatment of human immunodeficiency virus infection (Christeff et al., 1999; Lee et al., 1999). DHEA has been shown to protect mice from a variety of normally lethal infections. This includes protection against infection with bacteria (Ben-Nathan et al., 1999), and with parasites like Cryptosporidium parvum (Rasmussen et al., 1993, 1995), Plasmodium falciparum (Kurtis et al., 2001; Leenstra et al., 2003) and Schistosoma mansoni (Fallon et al., 1998; Morales-Montor et al., 2001).
DHEAS is a potent immune-activator modulating both T and B cell functions (Yang et al., 1998) and is responsible for augmenting antibody titers (Degelau et al., 1997). DHEAS also acts as a powerful down-modulator of the pro-inflammatory cytokines tumor necrosis factor-␣, interleukin-6, and interleukin1 (Danenberg et al., 1992; James et al., 1997), thus attenuating their deleterious consequences. A variety of biological activities of DHEA have been reported and include a decrease of total body weight (Aoki et al., 2004), an anti-diabetic effect with reduction of blood glucose tolerance (Coleman et al., 1982), and inhibition of lipid synthesis (Ben-David et al., 1967; Sonka et al., 1968; Kritchevsky et al., 1983). High DHEA or DHEAS levels have been suggested to be protective for cardiovascular disease (Khaw, 1996). DHEA supplementation is reported to lower levels of low-density cholesterol in humans (Nestler et al., 1992). To evaluate the response of male and female Wistar rats infected with the Y strain of T. cruzi treated with dehydroepiandrosterone we focus our analyses on its influences on parasitaemia, leucocytes, peritoneal macrophages, body weight and biochemical parameters such glucose, cholesterol and trygliceride levels.
2. Materials and methods 2.1. Animals and diet Male (n = 45) and female (n = 45) Wistar rats weighing 90–100 g were used. Animals were divided in groups: Male: Non-Infected Males-Without-DHEA treatment (MWDNI), Non-Infected Males-DHEA treated (MDNI), Infected Males-Without-DHEA treatment (MWDI), Infected Males-DHEA treated (MDI). Female: Non-Infected Females-Without-DHEA treatment (FWDNI), Non-Infected Females-DHEA treated (FDNI), Infected Females-Without-DHEA treatment (FWDI), Infected Females-DHEA treated (FDI). Rats were separated in number of 5 in plastic cages and commercial rodent diet and water were available ad libitum. Twelve hours before the experiment, animals were placed under dietary restriction and were given access to water only so as to minimize feeding-related variations in the biochemical parameters we measured.
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On the day of an experiment animals were weighed on an analytic balance. Rat pad was changed three times/week to avoid concentration of ammonia from urine. The protocol of this study was approved by the local Ethics Committee. 2.2. Infection and parasitaemia Rats were intraperitoneally (i.p.) inoculated with 1 × 105 blood tripomastigotes of the Y strain of T. cruzi (Silva and Nussenzweig, 1953). The experiments were performed on 7, 14 and 21 days after infection. Daily individual parasitaemias were determined by Brener’s method (Brener, 1962).
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infected male and female animals during the acute phase of infection. Blood sugar levels were determined by the glucose oxidase enzymatic method (Labtest, Minas Gerais, Brazil). Cholesterol was evaluated by the colorimetric oxidase method (Laborlab, S˜ao Paulo, Brazil) and tryglicerides by the reactive enzymatic method (Laborlab, S˜ao Paulo, Brazil). 2.8. Statistical analysis The results were expressed as the mean ± S.D. The one way ANOVA test was used to analyze data. Statistical significance between groups was determined by Tukey’s test. Differences were considered statistically significant when P < 0.05.
2.3. DHEA treatment Male and female rats were treated with 0.1 mL of DHEA (Sigma Chemical Co.), which had been dissolved in absolute ethanol (0.05 mL) and then diluted with an equal volume of distilled water (0.05 mL). DHEA was administered subcutaneously at a dose of 40 mg/kg body weight once a day over the course of the experiment. Treatment of the infected group started 48 h before infection. 2.4. Euthanasia Animals were decapitated with prior anesthesia using thribromoethanol 2.5%.
3. Results As shown in Fig. 1, the peak of parasitaemia occurred on 14 day after infection and on the 21 day post-infection parasites were absent. In untreated group, females displayed lesser number of parasites when compared to males (P < 0.001), the same occurred on infected group (P < 0.001). DHEA treatment caused an apparent reduction on parasitaemia levels in both male and female animals (P > 0.05), evidencing that DHEA triggered a decrease in parasite counts.
2.5. Leucocytes Leucocytes were evaluated using total blood with anticoagulant (EDTA 10%). Blood was diluted in Turkey’s solution and counted in Neubauer’s chamber. 2.6. Peritoneal macrophages Cells were collected from the peritoneal cavity, diluted in RPMI 1640 medium (Cultlab-Campinas Brazil), diluted with Turkey’s solution and counted in Neubauer chamber. 2.7. Biochemical analysis Glucose, cholesterol and tryglicerdes were determined in treated and non-treated, infected and non-
Fig. 1. Parasitaemia of male and female Wistar rats i.p. infected with 1 × 105 blood trypomastigotes of the Y strain of Trypanosoma cruzi, treated and non-treated with DHEA. Males-Without-DHEA treatment and Infected (MWDI), Males-DHEA treatment and Infected (MDI), Females-Without-DHEA treatment and Infected (FWDI), Females-DHEA treatment and Infected (FDI) (P < 0.05) n = 5 per group/day.
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Fig. 2. Leucocytes count of male and female Wistar rats non-infected and i.p. infected with 1 × 105 blood trypomastigotes of the Y strain of Trypanosoma cruzi, treated and non-treated with DHEA. Males-Without-DHEA treatment and Non-Infected (MWDNI), Males-DHEA treatment and Non-Infected (MDNI), Males-Without-DHEA treatment and Infected (MWDI), Males-DHEA treatment and Infected (MDI). Female: Females-Without-DHEA treatment and Non-Infected (FWDNI), Females-DHEA treatment and Non-Infected (FDNI), Females-Without-DHEA treatment and Infected (FWDI), Females-DHEA treatment and Infected (FDI) (P < 0.05) n = 5 per group/day.
Fig. 3. Peritoneal Macrophages count of male and female Wistar rats non-infected and i.p. infected with 1 × 105 blood trypomastigotes of the Y strain of Trypanosoma cruzi during the evolution of experimental disease. Males-Without-DHEA treatment and Non-Infected (MWDNI), Males-DHEA treatment and Non-Infected (MDNI), Males-Without-DHEA treatment and Infected (MWDI), Males-DHEA treatment and Infected (MDI). Female: Females-Without-DHEA treatment and Non-Infected (FWDNI), Females-DHEA treatment and Non-Infected (FDNI), FemalesWithout-DHEA treatment and Infected (FWDI), Females-DHEA treatment and Infected (FDI) (P < 0.0001) n = 5 per group/day.
Higher leucocytes values was observed in control females when compared to control males (P < 0.05). DHEA treatment did not triggered any significant alterations in leucocytes levels (P > 0.05) (Fig. 2). DHEA treatment was effective in enhancing macrophage number for male groups (P < 0.001) (Fig. 3). As shown in Table 1 untreated males displayed increased body weight compared to females. DHEA treatment triggered a non-significant lower weight in infected and non-infected males, while for females no significant alterations were observed. DHEA treatment induced a severe drop in blood glucose levels for all groups (P < 0.0001). For cholesterol
and tryglicerides no significant alterations (P > 0.05) were observed during DHEA treatment (Table 1).
4. Discussion Infectious diseases are still an important cause of mortality and morbidity and the urgent need to find an effective chemotherapy with fewer side effects and greater curative action would help the millions of people who are already infected. Because the currently available drugs have serious side-effects, the main goal of this work is to find an alternative treatment that improves the immune response through
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Table 1 Animal’s body weight, glucose, cholesterol and tryglicerides of male and female Wistar rats DHEA treated and non-treated, non-infected and i.p infected with 1 × 105 blood trypomastigotes of the Y strain of Trypanosoma cruzi during the evolution of experimental disease FWDNI FDNI FWDI FDI MWDNI MDNI MWDI MDI
Body Weight (g)
Glucose (mg/dl)
Cholesterol (md/dl)
Tryglicerides (mg/dl)
152 ± 8.5 159 ± 13.5 162 ± 13.7* 162 ± 14.7* 170 ± 10.4 159 ± 6.3 175 ± 8.9 160 ± 14.9
61 ± 9.7 26 ± 11.3* 58 ± 9.7 22 ± 9.3* 48 ± 7.1 11 ± 5.6* 72 ± 6.2 31 ± 5.4*
76 ± 22.7 71 ± 28.3 72 ± 13.9 66 ± 15.9 82 ± 22.5 73 ± 33.5 72 ± 23.5 59 ± 12.5
193 ± 76.2 191 ± 75.3 189 ± 64.4 185 ± 64.3 216 ± 49.2 203 ± 63.9 179 ± 40.7 180 ± 40.5
Comparison of Males-Without-DHEA treatment and Non-Infected (MWDNI), Males-DHEA treatment and Non-Infected (MDNI), MalesWithout-DHEA treatment and Infected (MWDI), Males-DHEA treatment and Infected (MDI). Female: Females-Without-DHEA treatment and Non-Infected (FWDNI), Females-DHEA treatment and Non-Infected (FDNI), Females-Without-DHEA treatment and Infected (FWDI), Females-DHEA treatment and Infected (FDI). ∗ Significant differences P < 0.0001.
the administration of an effective agent with low toxicity. The purpose of this study was to determine whether administration of dehydroepiandrosterone (DHEA) protects male and female rats during T. cruzi infection. The present study found that DHEA treatment enhances resistance for all animals to T. cruzi infection. Concerning to gender differences, our data confirms that females are more resistant to infection than males. Studies which evaluate cardiac function related to gender differences demonstrate that the process of myocardial adaptation after myocardial infarct differs significantly between male and female mice (Cavasin et al., 2004). Administration of exogenous DHEA has been demonstrated to increase the life-span of animals (Lucas et al., 1985) and to up-regulate the immune system (Loria et al., 1988). Anti-parasite activities of DHEA have been observed previously. DHEA supplementation reduces fecal Cryptosporidium parvum oocyst shedding and parasite colonization of the ilea of mice (Rasmussen et al., 1995). Another report shows that female mice treated with DHEAS were partially protected from Schistosoma mansoni infection (Fallon et al., 1998). The major finding in this report is an increased resistance of treated animals to reduce the blood parasites during acute phase of T. cruzi infection, mediated by the potent immune activating properties of this hormone suggesting a protective effect of DHEA in experimental T. cruzi infection.
DHEAS increases specific antibody responses (Abebe et al., 2003) and augments natural killer cell number and function (Morales-Montor et al., 2001). T. cruzi is a potent stimulator of cell-mediated immunity, and induction of macrophage pro-inflammatory cytokines is important for the control of infection and outcome in Chagas’ disease. Thus, the host’s resistance during experimental infection by T. cruzi is dependent on both innate and acquired immunity, that require the combined efforts of a number of cells including macrophages, natural killer cells, CD4+, and CD8+ T cells as well as antibody production by B cells (Tarleton et al., 1992; Brener and Gazzinelli, 1997). Since after DHEA administration, macrophage number was enhanced, we suggest that the protective effect can be due among other factors of macrophage activation leading to reduced parasitemia. Many animal and human studies show that supraphysiological doses of DHEA can influence body composition and carbohydrate and lipid metabolism. The biological activities of DHEA are: decrease in body weight and decrease of blood glucose (Coleman et al., 1982; Aoki et al., 2004). Much controversy has been aroused from some previous studies of DHEA’s effects because they were not randomized. In experiments with humans, the effects of treatment with 50 mg/24 h DHEA on the content and distribution of fat tissue and serum insulin, glucose, total cholesterol, low-density lipoprotein, cholesterol and high-density lipoprotein, cholesterol levels, as well as testosterone, estradiol, DHEA-S, prostate-
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specific antigen concentrations and indexes of insulin sensitivity and resistance were determined (Jedrzejuk et al., 2003). The results did not reveal any significant changes in the studied parameters, apart from a statistically significant increase in DHEA-S levels. However, in our work, significant reduction in the levels of glucose from rats infected and non-infected with T. cruzi, after DHEA administration was observed. Controversially with other works, cholesterol and tryglicerides did not show any statistically alteration during T. cruzi. Concerning to hormonal influences during experimental Chagas’ disease, corticosterone exerts an immunosuppressive effect, enhancing parasitaemia of male Wistar rats infected with the Y strain of T. cruzi, which underwent repetitive stress (Santos et al., 2005). Since glucocorticoids have a number of well-known anti-inflammatory effects and DHEA has a strong effect, increasing innate and adaptive immunity, and in addition, these products are unlikely to produce resistance, our studies provide a strong physiological basis for the development of new therapies based on adrenal steroid hormones. The strong effects of adrenal steroids upon T. cruzi may have implications in the development and treatment of Chagas’ disease enhancing the immune response probably being considered a starting point for a possible new alternative therapy drug.
Acknowledgments This study was supported by fellowships from CAPES and supported finance by FAPESP (03/063323). We thank Dr. S´ergio Akira Uyemura for the biochemistry analyses and David Peabody PhD (Medical School, University of New Mexico) for editorial assistance.
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Trypanosoma cruzi: The effects of dehydroepiandrosterone (DHEA) treatment during experimental infection Carla Domingues dos Santos ∗ , M´ıriam Paula Alonso Toldo, Jos´e Cl´ovis do Prado J´unior Laborat´orio de Parasitologia, Departamento de An´alises Cl´ınicas, Toxicol´ogicas e Bromatol´ogicas, Faculdade de Ciˆencias Farmacˆeuticas de Ribeir˜ao Preto FCFRP-USP, Universidade de S˜ao Paulo, Avenida do Caf´e s/no, 14040-903 Ribeir˜ao Preto, SP, Brazil Received 1 April 2005; received in revised form 7 April 2005; accepted 12 May 2005 Available online 13 June 2005
Abstract The aim of this study was to evaluate the efficacy of the immunomodulator dehydroepiandrosterone (DHEA) in the treatment of Trypanosoma cruzi infection and the possible biochemistry alterations in male and female Wistar rats. DHEA also known as the steroid of multiple actions has attracted distinct medical areas. Prior studies show that DHEA enhances immune responses against a wide range of viral, bacterial and parasitic pathogens. Furthermore, administration of DHEA seems to protect animals against obesity and diabetes. Male animals subcutaneous treated with 40 mg/kg body weight/day of DHEA displayed a significant reduction in blood parasites during parasitaemia peak, when compared to untreated animals (P < 0.001). For female group parasitaemia was also reduced although values are not statistically significant (P > 0.05). Sexual dimorphism was also observed, since females displayed lesser parasitaemia levels compared to males group treated (P > 0.05) and untreated (P < 0.001). Enhanced leucocytes number was observed in control females when compared to control males (P < 0.05). DHEA treatment did not triggered any significant alterations in leucocytes levels (P > 0.05). DHEA administration induced an enhanced number of macrophages in infected male (P < 0.01). DHEA administration causes a decrease in glucose (P < 0.001). Cholesterol and tryglicerides levels did not display results statistically significant (P > 0.05) during the treatment. These results suggest that DHEA treatment enhances the immune response as evidenced here by reduced levels of parasites. Up-regulation of the immune system by exogenous DHEA may be useful in the treatment of American tripanosomiasis. © 2005 Elsevier B.V. All rights reserved. Keywords: Dehydroepiandrosterone; Trypanosoma cruzi; Glucose; Peritoneal macrophages; Parasitemia; Leucocytes
1. Introduction ∗
Corresponding author. Tel.: +55 16 602 4153; fax: +55 16 602 4163. E-mail address: [email protected] (C.D. Santos). 0001-706X/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.actatropica.2005.05.005
Chagas’ disease is endemic in Latin America, affecting 16–18 million people, with more than 100 million exposed to the risk of infection (WHO, 1991).
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American trypanosomiasis is a protozoan infection caused by Trypanosoma cruzi and is one of the most important public health problems in Latin America. Gender and the corresponding sex steroids have shown several effects on immune response in a wide range of different species including humans, rodents and birds (Schuurs and Verheul, 1990). A bulk of evidence shows that sexual dimorphism plays an important role in the modulation of the immune response when hosts are exposed to distinct etiological agents. This means that a bi-directional relationship between endocrine and the immune system exists, in order to maintain homeostasis (Syrt and Sugarman, 1991; Prado et al., 1998, 1999). Women are more efficient in clearing peripheral parasitaemia these facts are probably linked to sexual dimorphism and steroid hormones (Brabin and Brabin, 1992). Estrogens exert a positive action on B cell proliferation, leading to enhanced immunoglobulin production. For this reason, women evade the pathological actions of a wide range of etiological agents more rapidly. Some of them produce severe illnesses in women such as Toxoplasma gondii and Trichomonas vaginalis (Roberts et al., 2001). DHEA is one of the steroid hormones produced by the adrenal cortex. DHEA sulphatase (DHEA-S) plays an important role in converting most all DHEA in DHEA sulphate (DHEAS), the latter form being one of the most common substances found in the circulation (Parker, 1995). Just before birth and again at puberty, DHEA starts to be produced in elevated concentrations, reaching its peak at around 20–30 years of age, followed by a progressive decline (Baulieu, 1996). DHEAS is also implicated in age-related changes in the immune system (Loria et al., 1988) and has been associated with disease susceptibility (Shealy, 1995). Currently, DHEA is one of numerous immunomodulators undergoing clinical evaluation as a potential treatment of human immunodeficiency virus infection (Christeff et al., 1999; Lee et al., 1999). DHEA has been shown to protect mice from a variety of normally lethal infections. This includes protection against infection with bacteria (Ben-Nathan et al., 1999), and with parasites like Cryptosporidium parvum (Rasmussen et al., 1993, 1995), Plasmodium falciparum (Kurtis et al., 2001; Leenstra et al., 2003) and Schistosoma mansoni (Fallon et al., 1998; Morales-Montor et al., 2001).
DHEAS is a potent immune-activator modulating both T and B cell functions (Yang et al., 1998) and is responsible for augmenting antibody titers (Degelau et al., 1997). DHEAS also acts as a powerful down-modulator of the pro-inflammatory cytokines tumor necrosis factor-␣, interleukin-6, and interleukin1 (Danenberg et al., 1992; James et al., 1997), thus attenuating their deleterious consequences. A variety of biological activities of DHEA have been reported and include a decrease of total body weight (Aoki et al., 2004), an anti-diabetic effect with reduction of blood glucose tolerance (Coleman et al., 1982), and inhibition of lipid synthesis (Ben-David et al., 1967; Sonka et al., 1968; Kritchevsky et al., 1983). High DHEA or DHEAS levels have been suggested to be protective for cardiovascular disease (Khaw, 1996). DHEA supplementation is reported to lower levels of low-density cholesterol in humans (Nestler et al., 1992). To evaluate the response of male and female Wistar rats infected with the Y strain of T. cruzi treated with dehydroepiandrosterone we focus our analyses on its influences on parasitaemia, leucocytes, peritoneal macrophages, body weight and biochemical parameters such glucose, cholesterol and trygliceride levels.
2. Materials and methods 2.1. Animals and diet Male (n = 45) and female (n = 45) Wistar rats weighing 90–100 g were used. Animals were divided in groups: Male: Non-Infected Males-Without-DHEA treatment (MWDNI), Non-Infected Males-DHEA treated (MDNI), Infected Males-Without-DHEA treatment (MWDI), Infected Males-DHEA treated (MDI). Female: Non-Infected Females-Without-DHEA treatment (FWDNI), Non-Infected Females-DHEA treated (FDNI), Infected Females-Without-DHEA treatment (FWDI), Infected Females-DHEA treated (FDI). Rats were separated in number of 5 in plastic cages and commercial rodent diet and water were available ad libitum. Twelve hours before the experiment, animals were placed under dietary restriction and were given access to water only so as to minimize feeding-related variations in the biochemical parameters we measured.
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On the day of an experiment animals were weighed on an analytic balance. Rat pad was changed three times/week to avoid concentration of ammonia from urine. The protocol of this study was approved by the local Ethics Committee. 2.2. Infection and parasitaemia Rats were intraperitoneally (i.p.) inoculated with 1 × 105 blood tripomastigotes of the Y strain of T. cruzi (Silva and Nussenzweig, 1953). The experiments were performed on 7, 14 and 21 days after infection. Daily individual parasitaemias were determined by Brener’s method (Brener, 1962).
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infected male and female animals during the acute phase of infection. Blood sugar levels were determined by the glucose oxidase enzymatic method (Labtest, Minas Gerais, Brazil). Cholesterol was evaluated by the colorimetric oxidase method (Laborlab, S˜ao Paulo, Brazil) and tryglicerides by the reactive enzymatic method (Laborlab, S˜ao Paulo, Brazil). 2.8. Statistical analysis The results were expressed as the mean ± S.D. The one way ANOVA test was used to analyze data. Statistical significance between groups was determined by Tukey’s test. Differences were considered statistically significant when P < 0.05.
2.3. DHEA treatment Male and female rats were treated with 0.1 mL of DHEA (Sigma Chemical Co.), which had been dissolved in absolute ethanol (0.05 mL) and then diluted with an equal volume of distilled water (0.05 mL). DHEA was administered subcutaneously at a dose of 40 mg/kg body weight once a day over the course of the experiment. Treatment of the infected group started 48 h before infection. 2.4. Euthanasia Animals were decapitated with prior anesthesia using thribromoethanol 2.5%.
3. Results As shown in Fig. 1, the peak of parasitaemia occurred on 14 day after infection and on the 21 day post-infection parasites were absent. In untreated group, females displayed lesser number of parasites when compared to males (P < 0.001), the same occurred on infected group (P < 0.001). DHEA treatment caused an apparent reduction on parasitaemia levels in both male and female animals (P > 0.05), evidencing that DHEA triggered a decrease in parasite counts.
2.5. Leucocytes Leucocytes were evaluated using total blood with anticoagulant (EDTA 10%). Blood was diluted in Turkey’s solution and counted in Neubauer’s chamber. 2.6. Peritoneal macrophages Cells were collected from the peritoneal cavity, diluted in RPMI 1640 medium (Cultlab-Campinas Brazil), diluted with Turkey’s solution and counted in Neubauer chamber. 2.7. Biochemical analysis Glucose, cholesterol and tryglicerdes were determined in treated and non-treated, infected and non-
Fig. 1. Parasitaemia of male and female Wistar rats i.p. infected with 1 × 105 blood trypomastigotes of the Y strain of Trypanosoma cruzi, treated and non-treated with DHEA. Males-Without-DHEA treatment and Infected (MWDI), Males-DHEA treatment and Infected (MDI), Females-Without-DHEA treatment and Infected (FWDI), Females-DHEA treatment and Infected (FDI) (P < 0.05) n = 5 per group/day.
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Fig. 2. Leucocytes count of male and female Wistar rats non-infected and i.p. infected with 1 × 105 blood trypomastigotes of the Y strain of Trypanosoma cruzi, treated and non-treated with DHEA. Males-Without-DHEA treatment and Non-Infected (MWDNI), Males-DHEA treatment and Non-Infected (MDNI), Males-Without-DHEA treatment and Infected (MWDI), Males-DHEA treatment and Infected (MDI). Female: Females-Without-DHEA treatment and Non-Infected (FWDNI), Females-DHEA treatment and Non-Infected (FDNI), Females-Without-DHEA treatment and Infected (FWDI), Females-DHEA treatment and Infected (FDI) (P < 0.05) n = 5 per group/day.
Fig. 3. Peritoneal Macrophages count of male and female Wistar rats non-infected and i.p. infected with 1 × 105 blood trypomastigotes of the Y strain of Trypanosoma cruzi during the evolution of experimental disease. Males-Without-DHEA treatment and Non-Infected (MWDNI), Males-DHEA treatment and Non-Infected (MDNI), Males-Without-DHEA treatment and Infected (MWDI), Males-DHEA treatment and Infected (MDI). Female: Females-Without-DHEA treatment and Non-Infected (FWDNI), Females-DHEA treatment and Non-Infected (FDNI), FemalesWithout-DHEA treatment and Infected (FWDI), Females-DHEA treatment and Infected (FDI) (P < 0.0001) n = 5 per group/day.
Higher leucocytes values was observed in control females when compared to control males (P < 0.05). DHEA treatment did not triggered any significant alterations in leucocytes levels (P > 0.05) (Fig. 2). DHEA treatment was effective in enhancing macrophage number for male groups (P < 0.001) (Fig. 3). As shown in Table 1 untreated males displayed increased body weight compared to females. DHEA treatment triggered a non-significant lower weight in infected and non-infected males, while for females no significant alterations were observed. DHEA treatment induced a severe drop in blood glucose levels for all groups (P < 0.0001). For cholesterol
and tryglicerides no significant alterations (P > 0.05) were observed during DHEA treatment (Table 1).
4. Discussion Infectious diseases are still an important cause of mortality and morbidity and the urgent need to find an effective chemotherapy with fewer side effects and greater curative action would help the millions of people who are already infected. Because the currently available drugs have serious side-effects, the main goal of this work is to find an alternative treatment that improves the immune response through
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Table 1 Animal’s body weight, glucose, cholesterol and tryglicerides of male and female Wistar rats DHEA treated and non-treated, non-infected and i.p infected with 1 × 105 blood trypomastigotes of the Y strain of Trypanosoma cruzi during the evolution of experimental disease FWDNI FDNI FWDI FDI MWDNI MDNI MWDI MDI
Body Weight (g)
Glucose (mg/dl)
Cholesterol (md/dl)
Tryglicerides (mg/dl)
152 ± 8.5 159 ± 13.5 162 ± 13.7* 162 ± 14.7* 170 ± 10.4 159 ± 6.3 175 ± 8.9 160 ± 14.9
61 ± 9.7 26 ± 11.3* 58 ± 9.7 22 ± 9.3* 48 ± 7.1 11 ± 5.6* 72 ± 6.2 31 ± 5.4*
76 ± 22.7 71 ± 28.3 72 ± 13.9 66 ± 15.9 82 ± 22.5 73 ± 33.5 72 ± 23.5 59 ± 12.5
193 ± 76.2 191 ± 75.3 189 ± 64.4 185 ± 64.3 216 ± 49.2 203 ± 63.9 179 ± 40.7 180 ± 40.5
Comparison of Males-Without-DHEA treatment and Non-Infected (MWDNI), Males-DHEA treatment and Non-Infected (MDNI), MalesWithout-DHEA treatment and Infected (MWDI), Males-DHEA treatment and Infected (MDI). Female: Females-Without-DHEA treatment and Non-Infected (FWDNI), Females-DHEA treatment and Non-Infected (FDNI), Females-Without-DHEA treatment and Infected (FWDI), Females-DHEA treatment and Infected (FDI). ∗ Significant differences P < 0.0001.
the administration of an effective agent with low toxicity. The purpose of this study was to determine whether administration of dehydroepiandrosterone (DHEA) protects male and female rats during T. cruzi infection. The present study found that DHEA treatment enhances resistance for all animals to T. cruzi infection. Concerning to gender differences, our data confirms that females are more resistant to infection than males. Studies which evaluate cardiac function related to gender differences demonstrate that the process of myocardial adaptation after myocardial infarct differs significantly between male and female mice (Cavasin et al., 2004). Administration of exogenous DHEA has been demonstrated to increase the life-span of animals (Lucas et al., 1985) and to up-regulate the immune system (Loria et al., 1988). Anti-parasite activities of DHEA have been observed previously. DHEA supplementation reduces fecal Cryptosporidium parvum oocyst shedding and parasite colonization of the ilea of mice (Rasmussen et al., 1995). Another report shows that female mice treated with DHEAS were partially protected from Schistosoma mansoni infection (Fallon et al., 1998). The major finding in this report is an increased resistance of treated animals to reduce the blood parasites during acute phase of T. cruzi infection, mediated by the potent immune activating properties of this hormone suggesting a protective effect of DHEA in experimental T. cruzi infection.
DHEAS increases specific antibody responses (Abebe et al., 2003) and augments natural killer cell number and function (Morales-Montor et al., 2001). T. cruzi is a potent stimulator of cell-mediated immunity, and induction of macrophage pro-inflammatory cytokines is important for the control of infection and outcome in Chagas’ disease. Thus, the host’s resistance during experimental infection by T. cruzi is dependent on both innate and acquired immunity, that require the combined efforts of a number of cells including macrophages, natural killer cells, CD4+, and CD8+ T cells as well as antibody production by B cells (Tarleton et al., 1992; Brener and Gazzinelli, 1997). Since after DHEA administration, macrophage number was enhanced, we suggest that the protective effect can be due among other factors of macrophage activation leading to reduced parasitemia. Many animal and human studies show that supraphysiological doses of DHEA can influence body composition and carbohydrate and lipid metabolism. The biological activities of DHEA are: decrease in body weight and decrease of blood glucose (Coleman et al., 1982; Aoki et al., 2004). Much controversy has been aroused from some previous studies of DHEA’s effects because they were not randomized. In experiments with humans, the effects of treatment with 50 mg/24 h DHEA on the content and distribution of fat tissue and serum insulin, glucose, total cholesterol, low-density lipoprotein, cholesterol and high-density lipoprotein, cholesterol levels, as well as testosterone, estradiol, DHEA-S, prostate-
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specific antigen concentrations and indexes of insulin sensitivity and resistance were determined (Jedrzejuk et al., 2003). The results did not reveal any significant changes in the studied parameters, apart from a statistically significant increase in DHEA-S levels. However, in our work, significant reduction in the levels of glucose from rats infected and non-infected with T. cruzi, after DHEA administration was observed. Controversially with other works, cholesterol and tryglicerides did not show any statistically alteration during T. cruzi. Concerning to hormonal influences during experimental Chagas’ disease, corticosterone exerts an immunosuppressive effect, enhancing parasitaemia of male Wistar rats infected with the Y strain of T. cruzi, which underwent repetitive stress (Santos et al., 2005). Since glucocorticoids have a number of well-known anti-inflammatory effects and DHEA has a strong effect, increasing innate and adaptive immunity, and in addition, these products are unlikely to produce resistance, our studies provide a strong physiological basis for the development of new therapies based on adrenal steroid hormones. The strong effects of adrenal steroids upon T. cruzi may have implications in the development and treatment of Chagas’ disease enhancing the immune response probably being considered a starting point for a possible new alternative therapy drug.
Acknowledgments This study was supported by fellowships from CAPES and supported finance by FAPESP (03/063323). We thank Dr. S´ergio Akira Uyemura for the biochemistry analyses and David Peabody PhD (Medical School, University of New Mexico) for editorial assistance.
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