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Trypanosoma cruzi: host selenium deficiency leads to higher mortality but similar parasitemia in mice
Experimental Parasitology 101 (2002) 193–199 www.elsevier.com/locate/yexpr
Trypanosoma cruzi: host selenium deficiency leads to higher mortality but similar parasitemia in mice Andrea P. de Souza,a Gabriel Melo de Oliveira,a Jean Neve,c Jean Vanderpas,d Claude Pirmez,b Solange L. de Castro,a Tania C. Ara ujo-Jorge,a,* and Maria Teresa Riveraa a
Lab. de Biologia Celular, Dept. de Ultra-estrutura e Biologia Celular, Instituto Oswaldo Cruz, Fundacßa~o Oswaldo Cruz, Av. Brasil 4365, Manguinhos, Rio de Janeiro, RJ 21045-900, Brazil b Lab. de Imunopatologia, Dept. de Bioquımica e Biologia Molecular, Instituto Oswaldo Cruz, Fundacßa~o Oswaldo Cruz, Av. Brasil 4365, Manguinhos, Rio de Janeiro, RJ 21045-900, Brazil c Fac. de Pharmacie, Univ. Libre de Bruxelles, Campus Plaine 205-5, Brussels B-1050, Belgium d H^ opital Ambroise-Par e, Bv. Kennedy 2, Mons 7000, Belgium Received 30 January 2002; received in revised form 6 August 2002; accepted 25 November 2002
Abstract Selenium is an essential trace element and its deficiency was implicated in heart diseases. We recently showed low Se levels in chronic chagasic patients with cardiomyopathy. Herein, mice were depleted in Se by feeding the mothers with chow containing only 0.005 mg Se/kg and maintaining this diet for offspring, that were further infected with Trypanosoma cruzi. Survival rate was significantly lower in Se deficient than in control mice. Parasitemia was similar in all groups. Necrotic heart lesions were found after infection (high CK-MB levels). No outbreaks of parasite growth were detected in chronic survivors submitted or not to a second Se depletion. The present results confirm our hypothesis that a nutritional deficiency in Se is associated to a higher mortality during T. cruzi infection. The potential beneficial effect of Se supplementation is a perspective. Hypothesis to explain the higher susceptibility of Se-depleted mice to T. cruzi infection are discussed. Index Descriptors and Abbreviations: Se, selenium; GPx (EC 1.11.1.9), glutathione peroxidade; CK-MB (EC 2.7.3.2), creatine kinase cardiac isoenzyme; Trypanosoma cruzi; ChagasÕ disease. Ó 2002 Elsevier Science (USA). All rights reserved.
1. Introduction Selenium (Se) is a trace element essential to organisms from bacteria to humans. One of its main role is an anti-oxidant action, involved in protection against damage caused by free radicals and oxidative stress (Diplock, 1987; Rayman, 2000). In mammals, the majority of Se is incorporated within protein as selenocysteine (reviewed in Stadtman, 1996). In mammals, one of the best characterized roles of Se is its incorporation in the active site of different isoforms of glutathione peroxidase (GPx), a fundamental enzyme for protection of polyunsaturated lipids in biological membranes; it catalyses the oxidation of reduced glutathione by hy*
Corresponding author. Fax: 55-21-2260-4434. E-mail address: taniaaj@ioc.fiocruz.br (T.C. Arau´jo-Jorge).
drogen peroxides or organic hydroperoxides (Rotruck et al., 1973). Se deficiency is implicated as a risk factor in cases of congestive myocardiopathy and in cardiovascular problems (Neve, 1996). In China Se deficiency is involved in the pathogenesis of Keshan disease, caused by a viral heart infection (Xu et al., 1997). Myocardiopathy is the most important feature in ChagasÕ disease, caused by Trypanosoma cruzi, which affects 13 million people in Latin American leading to high morbidity and mortality (WHO, 1997). In endemic areas, the population also suffers of malnutrition (Andrade and Zincker, 1995), but the impact of this factor on the course of human infection has been rarely studied. In a recent work with 180 chagasic chronic patients, we showed a decrease in Se serum levels in patients undergoing active myocardiopathy, which was positively correlated with ventricular
0014-4894/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved. doi:10.1016/S0014-4894(02)00134-0
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ejection fraction (Rivera et al., 2000, 2002). The association of Se deficiency to cardiopathies led us to investigate the influence of nutritional Se depletion in mice experimentally infected with T. cruzi and in the development of acute myocarditis. 2. Materials and methods 2.1. Selenium depletion Mice were depleted in selenium since the embryonic development, by feeding female C57BL/6 mice during pregnancy and lactation periods with chows containing different levels of Se (Hope Farms, Woerden, Holland), following a protocol developed for rats (Moreno-Reyes et al., 2001). Both chows have all the recommended nutrients, such as vitamins, amino acids (52.5 g/kg), oligoelements and fatty acids (41.2 g/kg), differing only in the Se levels: 0.2 mg Se/kg (control chow with adequate Se level), and 0.005 mg Se/kg (Se-deficient chow). After weaning (21 days after birth), the mothers [groups Cont Mo (n ¼ 11) and Se def Mo (n ¼ 12)] were sacrificed and the plasma and organs collected. The offsprings were separated by sex and submitted to the two different regimens throughout the experiment. 2.2. Infection, parasitemia, and mortality Offsprings aged 8–10 weeks were infected by intraperitoneal injection of 104 bloodstream forms of the Y strain (Silva and Nussenszweig, 1953) diluted in DulbeccoÕs modified EagleÕs medium. The groups were named Cont-f (females, n ¼ 9), Cont-m (males, n ¼ 11), Se def-f (females, n ¼ 8), and Se def-m (males, n ¼ 7). The level of parasitemia was checked by counting by the method Pizzi–Brener in 5 lL blood drawn from the tail of mice and diluted, when necessary, in 0.85% ammonium chloride. The mortality was noted daily and the following indices were calculated: cumulative mortality (percentage, %CM), survival time (ST) for succumbing mice expressed in days, and the day when mortality rate attained 50% ðM50 Þ. Surviving mice from the acute phase (n ¼ 11) were further monitored in the chronic phase 190 days post infection (dpi). They were divided in two groups, one receiving Se adequate diet (n ¼ 5) and the other receiving Se-deficient diet (n ¼ 6) for 2 months. The body weight was evaluated once a week and parasitemia levels were evaluated twice a week. Afterwards, they were sacrificed, the plasma collected for determination of CK-MB levels and the heart and liver were processed for histological analysis (see below). This work was performed in accordance with the guidelines established by the FIOCRUZ Commission of Ethics for the Use of Animals, resolution 099/99-PR.
2.3. Blood collection Approximately 30 lL blood were collected in heparinized micro-capillaries from the tail of each mouse prior to, and after different times during infection up to animal death or to 40 dpi. Plasma was obtained after centrifugation of blood in a micro-hematocrit centrifuge. Samples were frozen at )20 °C until analysis. 2.4. Determination of selenium levels The plasma levels of Se were measured by atomicabsorption spectrometry with the Zeeman background correction (model Z3030, Perkin–Elmer, Uberlingen, Germany) with a limit of sensitivity of 64 nM (5 ng/mL). Undetectable concentrations were assigned a value of 5 ng/mL (Moreno-Reyes et al., 1998; Neve et al., 1987). 2.5. Determination of GPx activity Glutathione peroxidase activity in the plasma was measured spectrophotometrically (k ¼ 340 nm) by the decrease in NADPH (0.28 mM) at 37 °C on a biochemical analyser (Hitachi 717, Boehringer–Mannheim, Mannhein, Germany), with an organic peroxide (isopropylbenzene [cumene] hydroperoxide; final concentration, 0.18 mM) and glutathione (final concentration, 4 mM) as substrates in 0.05 M of phosphate buffer (pH, 7.2) and 4.3 mM EDTA in the presence of excess glutathione reductase (P 0:05 M). The limit of sensitivity for the detection of plasma glutathione peroxidase was 50 U/L (Moreno-Reyes et al., 1998). 2.6. CK-MB assays The activity of CK-MB was measured before and after infection weekly, through commercial kit CK-MB Granutest (Merck Darmstadt, Germany). Incubation of plasma samples with the substrate leads to a net increase in NADPH concentration that is directly proportional to enzyme activity in the samples. The assay was adapted for reading in a microplate spectrophotometer (Spectramax 250 Molecular Device, USA), to allow the study of low volumes of mouse plasma (De Souza et al., 2000). Results were expressed in mean variation in optical densities obtained in each of six sequential readings at intervals of one minute each (DO.D./min). 2.7. Histopathological analysis Heart and liver tissues obtained from the groups Cont Mo and Se def Mo, as well as from chronic infected mice survivors from Cont-f and Cont-m, were processed in paraffin-embedded sections stained by hematoxylin-eosin. Sections were examined seeking histopathological alterations such as necrosis, fibrosis, inflammation, and/
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or the presence of parasites. Quantification of the histopathological alterations was performed under optical microscope by observation of 30 fields (objective 40) counting per area ðmm2 Þ the number of: (a) nests of parasites (cells with amastigotes); (b) foci of inflammatory infiltrate (containing at least 10 mononuclear cells). 2.8. Statistical analysis Statistical significance ðp < 0:05Þ was evaluated using Mann–Whitney non-parametric test for the parasitemia, Selenium levels, CK-MB and GPx activities. Survival analysis was plotted according to the Kaplan–Meier method. Survival rates in two groups of mice were compared with the log-rank test (Mantel-Cox), which follows a chi-square distribution with one degree of freedom.
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1994a,b; Vernet et al., 1999). At birth, we found a similar number of offsprings between females fed with both regimens, indicating that reproduction was not affected. CK-MB levels were normal and histopathological analysis showed absence of inflammatory infiltrate foci or necrosis in the heart of Se def Mo females (data not shown). Beck et al. (1994a) did also find no heart alteration in Se-depleted C3H/HeJ mice. Only one out of the 12 mothers evaluated in the Se-deficient group showed occasional and sparse foci of inflammatory infiltrate in the liver (data not shown). After the 21-day period of weaning, both female and male offsprings from the two groups of mothers showed normal motility, fur and skin conditions, bright eyes, similar weight (mean 8:4 0:2 g), and absence of clinical evidence of abnormalities. 3.2. Susceptibility of selenium-deficient mice to T. cruzi infection
3. Results and discussion 3.1. Nutritional selenium depletion Since Se is carried to the fetus via the placenta (Bedwal and Bahuguna, 1994) and to the newborn via the breast milk (Smith et al., 1982), Se deficiency was achieved by feeding 14 days pregnant female mice with Se 0.005 chow. GPx activity was detected in plasma as the Se status (Arthur, 1999). Measurements of Se levels and of GPx activity in the group Se def Mo confirmed the deficiency (Fig. 1), while Cont Mo had median levels of 214 ng Se/mL and 3933 U GPx/L, in Se def Mo the values were 40 ng Se/mL and 1130 U GPx/L, respectively. Thus, Se-deficient chow administered during mating, pregnancy and lactation led to a reduction of 5.4- and 3.5-, respectively of Se and GPx in the mothers, as reported by other authors (Beck et al.,
Given that the course of infection of C57BL/6 mice with the Y strain of T. cruzi is different between males and females (Souza et al., 2001), the data were presented by gender. Parasitemia curves presented the characteristic ascending and descending phases, attained similar maximal peaks and displayed similar kinetics and levels in groups Se-depleted or not, both in females and males mice (Fig. 2). The found no significant differences on the parasitemia among the four groups, thus suggesting that Se depletion did not interfere with parasite invasion into host cells or with its intracellular replication. This contrasts with infection by Coxsackie virus, since this pathogen replicates more intensely in nutritionally deficient host (Beck, 2000). Despite the similar parasitemia curves, mortality parameters clearly indicated that Se-deficient animals of both sexes were much more susceptible than the corre-
Fig. 1. Selenium levels (a) and GPx activity (b) from the groups of mothers Cont Mo and Se def Mo. Hatched bars represent median of control mothers (n ¼ 11), and white bars represent median of Se-deficient mothers (n ¼ 12).
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test was highly significant in both genders: 10.97 ðp ¼ 0:00093Þ in females and 8.03 ðp ¼ 0:00046Þ in males. The Se-deficient groups did not survive more than 23 days after infection, attaining 100% of mortality, while in the selenium adequate groups only 20% of the male and no female died even at 40 dpi. Since C57BL/6 mice are considered as a resistant strain to the high virulent Y strain of T. cruzi (Souza et al., 2001), the present results confirm our hypothesis that a nutritional deficiency in Se is associated to a higher mortality during T. cruzi infection. These findings also indicate that parasitemia and mortality are independent parameters, as already reported (De Titto, 1994; Wrightsman et al., 1982). 3.3. The mechanism of increased mortality could be immunological Fig. 2. Parasitemia curves in different groups of animals infected with T. cruzi: ðdÞ Cont-m (n ¼ 11); ðjÞ Cont-f (n ¼ 9); (s) Se def-m (n ¼ 7); ðÞ Se def-f (n ¼ 8).The results are expressed in median values.
sponding control groups (Table 1). Fig. 3 shows the survival rate according to time after infection with T. cruzi in the groups of female (Fig. 3a) or male (Fig. 3b) mice deficient or not in selenium. Comparing the controls and the Se-deficient groups, the log-rank statistic
Host depletion of Se is associated to a reduced immune response (reviewed in Beck, 2000), and an active immune response is essential to overcome the acute phase of T. cruzi infection. The difference found in mortality rate indicates that Se depletion interfered with innate and/or specific immune mechanisms that do operate in the host to control infection (the descending phase of parasitemia) and disease (the recover from acute phase). Since mice died 23 days after the infection
Table 1 Effect of selenium depletion on survival indexes of C57BL/6 mice infected with Trypanosoma cruzi Groups
Cont-f Se def-f Cont-m Se def-m a
Surviving time (days) Median
Percentile 25
Percentile 75
>40 19 >40 15
>40 13 >40 10
>40 22 >40 21
M50 a (days)
% Cumulative Mortality (40 dpi)
– 19 – 15
0 100 20 100
Day attaining 50% of mortality.
Fig. 3. Survival curves in different groups of animals infected with T. cruzi: (a) males ðdÞ Cont-m; ðsÞ Se def-m; (b) females ðjÞ Cont-f; ðÞ Se def-f.
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the mechanisms contributing tothe higher susceptibility of Se-depleted mice are likely to be related to an interference with the innate immune response. 3.4. Heart necrosis in selenium-deficient mice The markedly decrease in the survival of Se-depleted infected mice led us to investigate the presence of CKMB in plasma, as a parameter of heart necrosis (Alpert et al., 2000; De Souza et al., 2000). In a kinetic study using this model, we have shown that the peak of heart necrotic lesion occurs 15 days after infection, and that infected mice recover from acute myocarditis (Henriques-Pons et al., 2002). Since most Se-deficient male mice did not surpass 15 dpi, only female mice were analyzed. CK-MB levels increased significantly with the development of infection in both groups (Fig. 4). In Cont-f, median values were 0.016 and 0.045 DO.D./min, respectively before and 15 days after infection. The corresponding values for Se def-f were 0.017 and 0.113 DO.D./min. In the Cont-f group, almost all mice (4 out of 5) presented a high increase in CK-MB levels (up to 7:7), but recovered and survived the acute phase. All the infected mice in Se def-f group died, despite a similar range of increase (2.6 to 7:4) in plasma CK-MB activity (Fig. 4). A single animal in the control group did
Fig. 4. CK-MB plasma levels in individual mice from Cont-f (n ¼ 5) and Se def-f (n ¼ 5). Lines link results from a same animal. Arrows indicate significant differences (p < 0:05) of CK-MB levels of the group before (d0) and 15 days after infection (d15); number indicate the increase as compared to the basal level of each mice before infection; the letters s and d indicate, respectively, mice that survived and died during the acute phase.
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not show myocardial damage (decrease in CK-MB), but it was not monitored later for a conclusive interpretation of this result. This indicated that both groups presented necrotic heart lesions after T. cruzi infection, as shown by Mercado and Garbus (1976, 1979), but that only the animals receiving normal Se chow were able to recover from acute myocarditis. Under viral infections, it was reported that Se deficiency led to heart necrosis (Beck et al., 1994a,b). Because all the mice from the Sedeficient groups died we could not follow if statistically significant differences between the groups submitted to control and Se-deficient diets would occur after the 15th dpi. Therefore, although Se deficiency strongly contributed to the mortality of the animals, we cannot presently associate heart damage with death. Our results suggest that other mechanisms are involved in the higher susceptibility of mice fed with the chow containing only 0.005 mg/kg Se. The survivors of group Cont-f were followed up to the 75th dpi, and CK-MB levels were found to be high (data not shown), suggesting that an adequate Se diet does not prevent myocardial damage in mice challenged with T. cruzi. 3.5. Effect of selenium depletion during the chronic phase of infection The same survivors (n ¼ 11) in whom we studied CKMB levels were further submitted to selenium depletion after 190 dpi, by feeding 6 mice with deficient Se chow. No alterations were observed in the body weight and the parasitemia was maintained at sub-patent levels (data not shown). After 2 months, mice were sacrificed and analyzed. No histopathological heart alteration was observed in any mice from both groups (data not shown). It is known that during the chronic phase, immunosuppressive agents may lead to outbreaks of parasite growth, with patent parasitemia, both in humans (Pizzi et al., 1982) and in experimental animals (Calabrese et al., 1996). In addition, no amastigote nests were found in the heart of any mice, suggesting that an overall immune suppression was probably not induced by nutritional deficiency of Se. T. cruzi infection is associated with an acute and transient immunosuppression characterized by a decrease in both humoral and cellular responses (reviewed by Tarleton, 1991), with a high polyclonal activation (Reina-San-Martin et al., 2000). It is thus possible that Se deficiency could exacerbate the impairment of both responses in infected mice fed with Se-deficient chow. Beck et al. (1994a) showed that lymphocyte proliferation to mitogen or antigens decreased in Se-deficient mice infected with a cardiovirulent virus strain. It is possible that Se-deficient mice could present low activity of T CD8þ cells, pivotal cells for the protective response against T. cruzi infection (Sun and Tarleton, 1993). Selenium was also shown to be important for the
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expression of high affinity IL-2 receptors in T cells (Roy et al., 1993), a mechanism that can also concur to the higher susceptibility presented by Se-deficient mice, since down modulation of IL2-R was verified in T. cruzi infection both in humans (Reis et al., 1997) and in mice (Majumder and Kierszenbaum, 1996). A key perspective of the present study, associated to the findings of lower Se levels in chronic chagasic patients with active myocardiopathy (Rivera et al., 2000), is the potential beneficial effect of Se supplementation. This matter was recently reviewed by Neve (2000) and experimental and human protocols for monitoring the evolution of chronic experimental myocardiopathy under selenium supplementation are underway in our laboratory.
Acknowledgments We are grateful to Dr. Linda Jelicks for critically reviewing the manuscript and to Marcos Meuser Baptista and to Alexandre H. de Oliveira for their excellent technical assistance. This work was supported with grants from CNPq, PAPES/FIOCRUZ and FAPERJ.
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Trypanosoma cruzi: host selenium deficiency leads to higher mortality but similar parasitemia in mice Andrea P. de Souza,a Gabriel Melo de Oliveira,a Jean Neve,c Jean Vanderpas,d Claude Pirmez,b Solange L. de Castro,a Tania C. Ara ujo-Jorge,a,* and Maria Teresa Riveraa a
Lab. de Biologia Celular, Dept. de Ultra-estrutura e Biologia Celular, Instituto Oswaldo Cruz, Fundacßa~o Oswaldo Cruz, Av. Brasil 4365, Manguinhos, Rio de Janeiro, RJ 21045-900, Brazil b Lab. de Imunopatologia, Dept. de Bioquımica e Biologia Molecular, Instituto Oswaldo Cruz, Fundacßa~o Oswaldo Cruz, Av. Brasil 4365, Manguinhos, Rio de Janeiro, RJ 21045-900, Brazil c Fac. de Pharmacie, Univ. Libre de Bruxelles, Campus Plaine 205-5, Brussels B-1050, Belgium d H^ opital Ambroise-Par e, Bv. Kennedy 2, Mons 7000, Belgium Received 30 January 2002; received in revised form 6 August 2002; accepted 25 November 2002
Abstract Selenium is an essential trace element and its deficiency was implicated in heart diseases. We recently showed low Se levels in chronic chagasic patients with cardiomyopathy. Herein, mice were depleted in Se by feeding the mothers with chow containing only 0.005 mg Se/kg and maintaining this diet for offspring, that were further infected with Trypanosoma cruzi. Survival rate was significantly lower in Se deficient than in control mice. Parasitemia was similar in all groups. Necrotic heart lesions were found after infection (high CK-MB levels). No outbreaks of parasite growth were detected in chronic survivors submitted or not to a second Se depletion. The present results confirm our hypothesis that a nutritional deficiency in Se is associated to a higher mortality during T. cruzi infection. The potential beneficial effect of Se supplementation is a perspective. Hypothesis to explain the higher susceptibility of Se-depleted mice to T. cruzi infection are discussed. Index Descriptors and Abbreviations: Se, selenium; GPx (EC 1.11.1.9), glutathione peroxidade; CK-MB (EC 2.7.3.2), creatine kinase cardiac isoenzyme; Trypanosoma cruzi; ChagasÕ disease. Ó 2002 Elsevier Science (USA). All rights reserved.
1. Introduction Selenium (Se) is a trace element essential to organisms from bacteria to humans. One of its main role is an anti-oxidant action, involved in protection against damage caused by free radicals and oxidative stress (Diplock, 1987; Rayman, 2000). In mammals, the majority of Se is incorporated within protein as selenocysteine (reviewed in Stadtman, 1996). In mammals, one of the best characterized roles of Se is its incorporation in the active site of different isoforms of glutathione peroxidase (GPx), a fundamental enzyme for protection of polyunsaturated lipids in biological membranes; it catalyses the oxidation of reduced glutathione by hy*
Corresponding author. Fax: 55-21-2260-4434. E-mail address: taniaaj@ioc.fiocruz.br (T.C. Arau´jo-Jorge).
drogen peroxides or organic hydroperoxides (Rotruck et al., 1973). Se deficiency is implicated as a risk factor in cases of congestive myocardiopathy and in cardiovascular problems (Neve, 1996). In China Se deficiency is involved in the pathogenesis of Keshan disease, caused by a viral heart infection (Xu et al., 1997). Myocardiopathy is the most important feature in ChagasÕ disease, caused by Trypanosoma cruzi, which affects 13 million people in Latin American leading to high morbidity and mortality (WHO, 1997). In endemic areas, the population also suffers of malnutrition (Andrade and Zincker, 1995), but the impact of this factor on the course of human infection has been rarely studied. In a recent work with 180 chagasic chronic patients, we showed a decrease in Se serum levels in patients undergoing active myocardiopathy, which was positively correlated with ventricular
0014-4894/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved. doi:10.1016/S0014-4894(02)00134-0
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ejection fraction (Rivera et al., 2000, 2002). The association of Se deficiency to cardiopathies led us to investigate the influence of nutritional Se depletion in mice experimentally infected with T. cruzi and in the development of acute myocarditis. 2. Materials and methods 2.1. Selenium depletion Mice were depleted in selenium since the embryonic development, by feeding female C57BL/6 mice during pregnancy and lactation periods with chows containing different levels of Se (Hope Farms, Woerden, Holland), following a protocol developed for rats (Moreno-Reyes et al., 2001). Both chows have all the recommended nutrients, such as vitamins, amino acids (52.5 g/kg), oligoelements and fatty acids (41.2 g/kg), differing only in the Se levels: 0.2 mg Se/kg (control chow with adequate Se level), and 0.005 mg Se/kg (Se-deficient chow). After weaning (21 days after birth), the mothers [groups Cont Mo (n ¼ 11) and Se def Mo (n ¼ 12)] were sacrificed and the plasma and organs collected. The offsprings were separated by sex and submitted to the two different regimens throughout the experiment. 2.2. Infection, parasitemia, and mortality Offsprings aged 8–10 weeks were infected by intraperitoneal injection of 104 bloodstream forms of the Y strain (Silva and Nussenszweig, 1953) diluted in DulbeccoÕs modified EagleÕs medium. The groups were named Cont-f (females, n ¼ 9), Cont-m (males, n ¼ 11), Se def-f (females, n ¼ 8), and Se def-m (males, n ¼ 7). The level of parasitemia was checked by counting by the method Pizzi–Brener in 5 lL blood drawn from the tail of mice and diluted, when necessary, in 0.85% ammonium chloride. The mortality was noted daily and the following indices were calculated: cumulative mortality (percentage, %CM), survival time (ST) for succumbing mice expressed in days, and the day when mortality rate attained 50% ðM50 Þ. Surviving mice from the acute phase (n ¼ 11) were further monitored in the chronic phase 190 days post infection (dpi). They were divided in two groups, one receiving Se adequate diet (n ¼ 5) and the other receiving Se-deficient diet (n ¼ 6) for 2 months. The body weight was evaluated once a week and parasitemia levels were evaluated twice a week. Afterwards, they were sacrificed, the plasma collected for determination of CK-MB levels and the heart and liver were processed for histological analysis (see below). This work was performed in accordance with the guidelines established by the FIOCRUZ Commission of Ethics for the Use of Animals, resolution 099/99-PR.
2.3. Blood collection Approximately 30 lL blood were collected in heparinized micro-capillaries from the tail of each mouse prior to, and after different times during infection up to animal death or to 40 dpi. Plasma was obtained after centrifugation of blood in a micro-hematocrit centrifuge. Samples were frozen at )20 °C until analysis. 2.4. Determination of selenium levels The plasma levels of Se were measured by atomicabsorption spectrometry with the Zeeman background correction (model Z3030, Perkin–Elmer, Uberlingen, Germany) with a limit of sensitivity of 64 nM (5 ng/mL). Undetectable concentrations were assigned a value of 5 ng/mL (Moreno-Reyes et al., 1998; Neve et al., 1987). 2.5. Determination of GPx activity Glutathione peroxidase activity in the plasma was measured spectrophotometrically (k ¼ 340 nm) by the decrease in NADPH (0.28 mM) at 37 °C on a biochemical analyser (Hitachi 717, Boehringer–Mannheim, Mannhein, Germany), with an organic peroxide (isopropylbenzene [cumene] hydroperoxide; final concentration, 0.18 mM) and glutathione (final concentration, 4 mM) as substrates in 0.05 M of phosphate buffer (pH, 7.2) and 4.3 mM EDTA in the presence of excess glutathione reductase (P 0:05 M). The limit of sensitivity for the detection of plasma glutathione peroxidase was 50 U/L (Moreno-Reyes et al., 1998). 2.6. CK-MB assays The activity of CK-MB was measured before and after infection weekly, through commercial kit CK-MB Granutest (Merck Darmstadt, Germany). Incubation of plasma samples with the substrate leads to a net increase in NADPH concentration that is directly proportional to enzyme activity in the samples. The assay was adapted for reading in a microplate spectrophotometer (Spectramax 250 Molecular Device, USA), to allow the study of low volumes of mouse plasma (De Souza et al., 2000). Results were expressed in mean variation in optical densities obtained in each of six sequential readings at intervals of one minute each (DO.D./min). 2.7. Histopathological analysis Heart and liver tissues obtained from the groups Cont Mo and Se def Mo, as well as from chronic infected mice survivors from Cont-f and Cont-m, were processed in paraffin-embedded sections stained by hematoxylin-eosin. Sections were examined seeking histopathological alterations such as necrosis, fibrosis, inflammation, and/
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or the presence of parasites. Quantification of the histopathological alterations was performed under optical microscope by observation of 30 fields (objective 40) counting per area ðmm2 Þ the number of: (a) nests of parasites (cells with amastigotes); (b) foci of inflammatory infiltrate (containing at least 10 mononuclear cells). 2.8. Statistical analysis Statistical significance ðp < 0:05Þ was evaluated using Mann–Whitney non-parametric test for the parasitemia, Selenium levels, CK-MB and GPx activities. Survival analysis was plotted according to the Kaplan–Meier method. Survival rates in two groups of mice were compared with the log-rank test (Mantel-Cox), which follows a chi-square distribution with one degree of freedom.
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1994a,b; Vernet et al., 1999). At birth, we found a similar number of offsprings between females fed with both regimens, indicating that reproduction was not affected. CK-MB levels were normal and histopathological analysis showed absence of inflammatory infiltrate foci or necrosis in the heart of Se def Mo females (data not shown). Beck et al. (1994a) did also find no heart alteration in Se-depleted C3H/HeJ mice. Only one out of the 12 mothers evaluated in the Se-deficient group showed occasional and sparse foci of inflammatory infiltrate in the liver (data not shown). After the 21-day period of weaning, both female and male offsprings from the two groups of mothers showed normal motility, fur and skin conditions, bright eyes, similar weight (mean 8:4 0:2 g), and absence of clinical evidence of abnormalities. 3.2. Susceptibility of selenium-deficient mice to T. cruzi infection
3. Results and discussion 3.1. Nutritional selenium depletion Since Se is carried to the fetus via the placenta (Bedwal and Bahuguna, 1994) and to the newborn via the breast milk (Smith et al., 1982), Se deficiency was achieved by feeding 14 days pregnant female mice with Se 0.005 chow. GPx activity was detected in plasma as the Se status (Arthur, 1999). Measurements of Se levels and of GPx activity in the group Se def Mo confirmed the deficiency (Fig. 1), while Cont Mo had median levels of 214 ng Se/mL and 3933 U GPx/L, in Se def Mo the values were 40 ng Se/mL and 1130 U GPx/L, respectively. Thus, Se-deficient chow administered during mating, pregnancy and lactation led to a reduction of 5.4- and 3.5-, respectively of Se and GPx in the mothers, as reported by other authors (Beck et al.,
Given that the course of infection of C57BL/6 mice with the Y strain of T. cruzi is different between males and females (Souza et al., 2001), the data were presented by gender. Parasitemia curves presented the characteristic ascending and descending phases, attained similar maximal peaks and displayed similar kinetics and levels in groups Se-depleted or not, both in females and males mice (Fig. 2). The found no significant differences on the parasitemia among the four groups, thus suggesting that Se depletion did not interfere with parasite invasion into host cells or with its intracellular replication. This contrasts with infection by Coxsackie virus, since this pathogen replicates more intensely in nutritionally deficient host (Beck, 2000). Despite the similar parasitemia curves, mortality parameters clearly indicated that Se-deficient animals of both sexes were much more susceptible than the corre-
Fig. 1. Selenium levels (a) and GPx activity (b) from the groups of mothers Cont Mo and Se def Mo. Hatched bars represent median of control mothers (n ¼ 11), and white bars represent median of Se-deficient mothers (n ¼ 12).
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test was highly significant in both genders: 10.97 ðp ¼ 0:00093Þ in females and 8.03 ðp ¼ 0:00046Þ in males. The Se-deficient groups did not survive more than 23 days after infection, attaining 100% of mortality, while in the selenium adequate groups only 20% of the male and no female died even at 40 dpi. Since C57BL/6 mice are considered as a resistant strain to the high virulent Y strain of T. cruzi (Souza et al., 2001), the present results confirm our hypothesis that a nutritional deficiency in Se is associated to a higher mortality during T. cruzi infection. These findings also indicate that parasitemia and mortality are independent parameters, as already reported (De Titto, 1994; Wrightsman et al., 1982). 3.3. The mechanism of increased mortality could be immunological Fig. 2. Parasitemia curves in different groups of animals infected with T. cruzi: ðdÞ Cont-m (n ¼ 11); ðjÞ Cont-f (n ¼ 9); (s) Se def-m (n ¼ 7); ðÞ Se def-f (n ¼ 8).The results are expressed in median values.
sponding control groups (Table 1). Fig. 3 shows the survival rate according to time after infection with T. cruzi in the groups of female (Fig. 3a) or male (Fig. 3b) mice deficient or not in selenium. Comparing the controls and the Se-deficient groups, the log-rank statistic
Host depletion of Se is associated to a reduced immune response (reviewed in Beck, 2000), and an active immune response is essential to overcome the acute phase of T. cruzi infection. The difference found in mortality rate indicates that Se depletion interfered with innate and/or specific immune mechanisms that do operate in the host to control infection (the descending phase of parasitemia) and disease (the recover from acute phase). Since mice died 23 days after the infection
Table 1 Effect of selenium depletion on survival indexes of C57BL/6 mice infected with Trypanosoma cruzi Groups
Cont-f Se def-f Cont-m Se def-m a
Surviving time (days) Median
Percentile 25
Percentile 75
>40 19 >40 15
>40 13 >40 10
>40 22 >40 21
M50 a (days)
% Cumulative Mortality (40 dpi)
– 19 – 15
0 100 20 100
Day attaining 50% of mortality.
Fig. 3. Survival curves in different groups of animals infected with T. cruzi: (a) males ðdÞ Cont-m; ðsÞ Se def-m; (b) females ðjÞ Cont-f; ðÞ Se def-f.
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the mechanisms contributing tothe higher susceptibility of Se-depleted mice are likely to be related to an interference with the innate immune response. 3.4. Heart necrosis in selenium-deficient mice The markedly decrease in the survival of Se-depleted infected mice led us to investigate the presence of CKMB in plasma, as a parameter of heart necrosis (Alpert et al., 2000; De Souza et al., 2000). In a kinetic study using this model, we have shown that the peak of heart necrotic lesion occurs 15 days after infection, and that infected mice recover from acute myocarditis (Henriques-Pons et al., 2002). Since most Se-deficient male mice did not surpass 15 dpi, only female mice were analyzed. CK-MB levels increased significantly with the development of infection in both groups (Fig. 4). In Cont-f, median values were 0.016 and 0.045 DO.D./min, respectively before and 15 days after infection. The corresponding values for Se def-f were 0.017 and 0.113 DO.D./min. In the Cont-f group, almost all mice (4 out of 5) presented a high increase in CK-MB levels (up to 7:7), but recovered and survived the acute phase. All the infected mice in Se def-f group died, despite a similar range of increase (2.6 to 7:4) in plasma CK-MB activity (Fig. 4). A single animal in the control group did
Fig. 4. CK-MB plasma levels in individual mice from Cont-f (n ¼ 5) and Se def-f (n ¼ 5). Lines link results from a same animal. Arrows indicate significant differences (p < 0:05) of CK-MB levels of the group before (d0) and 15 days after infection (d15); number indicate the increase as compared to the basal level of each mice before infection; the letters s and d indicate, respectively, mice that survived and died during the acute phase.
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not show myocardial damage (decrease in CK-MB), but it was not monitored later for a conclusive interpretation of this result. This indicated that both groups presented necrotic heart lesions after T. cruzi infection, as shown by Mercado and Garbus (1976, 1979), but that only the animals receiving normal Se chow were able to recover from acute myocarditis. Under viral infections, it was reported that Se deficiency led to heart necrosis (Beck et al., 1994a,b). Because all the mice from the Sedeficient groups died we could not follow if statistically significant differences between the groups submitted to control and Se-deficient diets would occur after the 15th dpi. Therefore, although Se deficiency strongly contributed to the mortality of the animals, we cannot presently associate heart damage with death. Our results suggest that other mechanisms are involved in the higher susceptibility of mice fed with the chow containing only 0.005 mg/kg Se. The survivors of group Cont-f were followed up to the 75th dpi, and CK-MB levels were found to be high (data not shown), suggesting that an adequate Se diet does not prevent myocardial damage in mice challenged with T. cruzi. 3.5. Effect of selenium depletion during the chronic phase of infection The same survivors (n ¼ 11) in whom we studied CKMB levels were further submitted to selenium depletion after 190 dpi, by feeding 6 mice with deficient Se chow. No alterations were observed in the body weight and the parasitemia was maintained at sub-patent levels (data not shown). After 2 months, mice were sacrificed and analyzed. No histopathological heart alteration was observed in any mice from both groups (data not shown). It is known that during the chronic phase, immunosuppressive agents may lead to outbreaks of parasite growth, with patent parasitemia, both in humans (Pizzi et al., 1982) and in experimental animals (Calabrese et al., 1996). In addition, no amastigote nests were found in the heart of any mice, suggesting that an overall immune suppression was probably not induced by nutritional deficiency of Se. T. cruzi infection is associated with an acute and transient immunosuppression characterized by a decrease in both humoral and cellular responses (reviewed by Tarleton, 1991), with a high polyclonal activation (Reina-San-Martin et al., 2000). It is thus possible that Se deficiency could exacerbate the impairment of both responses in infected mice fed with Se-deficient chow. Beck et al. (1994a) showed that lymphocyte proliferation to mitogen or antigens decreased in Se-deficient mice infected with a cardiovirulent virus strain. It is possible that Se-deficient mice could present low activity of T CD8þ cells, pivotal cells for the protective response against T. cruzi infection (Sun and Tarleton, 1993). Selenium was also shown to be important for the
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expression of high affinity IL-2 receptors in T cells (Roy et al., 1993), a mechanism that can also concur to the higher susceptibility presented by Se-deficient mice, since down modulation of IL2-R was verified in T. cruzi infection both in humans (Reis et al., 1997) and in mice (Majumder and Kierszenbaum, 1996). A key perspective of the present study, associated to the findings of lower Se levels in chronic chagasic patients with active myocardiopathy (Rivera et al., 2000), is the potential beneficial effect of Se supplementation. This matter was recently reviewed by Neve (2000) and experimental and human protocols for monitoring the evolution of chronic experimental myocardiopathy under selenium supplementation are underway in our laboratory.
Acknowledgments We are grateful to Dr. Linda Jelicks for critically reviewing the manuscript and to Marcos Meuser Baptista and to Alexandre H. de Oliveira for their excellent technical assistance. This work was supported with grants from CNPq, PAPES/FIOCRUZ and FAPERJ.
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