Experimental Parasitology 147 (2014) 1–6
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Effects of iron supplementation on blood adenine deaminase activity and oxidative stress in Trypanosoma evansi infection of rats Nathieli B. Bottari a,b, Matheus D. Baldissera a, Alexandre A. Tonin a, Raqueli T. França c, Danieli Zanini b, Marta L.R. Leal d, Sonia T.A. Lopes c, Maria Rosa C. Schetinger b, Vera M. Morsch b, Silvia G. Monteiro a, Naiara S. Guarda f, Rafael N. Moresco f, Adelina R. Aires d, Lenita M. Stefani e, Aleksandro S. Da Silva b,e,* a
Department of Microbiology and Parasitology, Universidade Federal de Santa Maria (UFSM), Santa Maria, RS, Brazil Toxicological Biochemistry of Graduate Program, UFSM, Santa Maria, RS, Brazil c Department of Small Animal, UFSM, Santa Maria, RS, Brazil d Department of Large Animal, UFSM, Santa Maria, RS, Brazil e Department of Animal Science, Universidade do Estado de Santa Catarina (UDESC), Chapecó, SC, Brazil f Department of Clinical and Toxicological Analysis, UFSM, Santa Maria, RS, Brazil b
H I G H L I G H T
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G R A P H I C A L
Trypanosoma evansi is the etiological agent of the disease known as “Surra” or “Mal das cadeiras”. T. evansi infection plays an important role in the anemia, iron levels, ADA activity and oxidative stress. Iron supplementation was able to provide some effect on the oxidative stress imbalance, as well as in the ADA modulation. Iron supplementation helped to diminish the parasitemia, and indirectly reduced the anemic process.
A R T I C L E
I N F O
Article history: Received 16 April 2014 Received in revised form 11 July 2014 Accepted 15 September 2014 Available online 6 October 2014 Keywords: Trypanosomosis Anemia ADA Oxidants Antioxidants
Infected by Trypanosoma evansi – day 0
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and Iron supplementation at days 1 and 6 postinfection
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Supplementation was able to provide some effect on the oxidative stress imbalance and anemia, as well as in the ADA activity modulation
A B S T R A C T
The aim of this study was to assess the effects of iron supplementation on oxidative stress and on the activity of the adenosine deaminase (ADA) in rats experimentally infected by Trypanosoma evansi. For this purpose, 20 rats were divided into four experimental groups with five animals each as follows: groups A and B were composed by healthy animals, while animals from groups C and D were infected by T. evansi. Additionally, groups B and D received two subcutaneous doses of iron (60 mg kg-1) within an interval of 5 days. Blood samples were drawn on day 8 post infection in order to assess hematological and biochemical variables. Among the main results are: (1) animals from group C showed reduced erythrogram (with tendency to anemia); however the same results were not observed for group D; this might be a direct effect of free iron on trypanosomes which helped to reduce the parasitemia and the damage to erythrocytes caused by the infection; (2) iron supplementation was able to reduce NOx levels by inhibiting iNOS, and thus, providing an antioxidant action and, indirectly, reducing the ALT levels in groups B
* Corresponding author. Fax: +55 49 3330-9440. E-mail address: [email protected] (A.S. Da Silva). http://dx.doi.org/10.1016/j.exppara.2014.09.002 0014-4894/© 2014 Elsevier Inc. All rights reserved.
A B S T R A C T
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and D; (3) increase FRAP levels in group D; (4) reduce ADA activity in serum and erythrocytes in group C; however, this supplementation (5) increased the protein oxidation in groups B and D, as well as group C (positive control). Therefore, iron showed antioxidant and oxidant effects on animals that received supplementation; and it maintained the activity of E-ADA stable in infected/supplemented animals. © 2014 Elsevier Inc. All rights reserved.
1. Introduction Trypanosoma evansi is the etiological agent of the disease known as “Surra” or “Mal das cadeiras” in horses. It shows a wide geographical distribution, being found parasitizing various species of domestic and wild animals (Silva et al., 2002). Rapid weight loss, anemia, swollen limbs, progressive weakness, neurological signs and paralysis of the hind limbs (in chronic cases) are the clinical signs usually observed in trypanosomosis (Herrera et al., 2004; Rodrigues et al., 2005). The disease in its acute form is characterized by hematological changes, progressive anemia, anorexia, leading in some cases to death (Omer et al., 2007). T. evansi infection is able to cause an inflammatory response in the host (Paim et al., 2011). Considering that inflammation is a stereotypical physiological response to infections and tissue injury, which leads to pathogen killing as well as tissue repair processes, helping to restore homeostasis at infected or damaged sites (Calder et al., 2009), it is important to consider on this conditions, may changes in the plasma level of certain trace metals (e.g., a reduction in plasma iron) can be observed (Weinberg, 1978). Iron is an essential nutrient for biochemical processes, such as electron transfer and regulation of cell growth, transport oxygen. It is also essential for cellular homeostasis, DNA synthesis, energetic metabolism and synthesis of heme group (Iannotti et al., 2006; Smith, 2002). Iron redox enzymes are essential for cell respiration, DNA synthesis, and free radical-scavenging mechanisms, but in the presence of an inflammatory process, we observe a lack of free iron (Doherty, 2007). According to Wolkmer et al. (2007) and Da Silva et al. (2009) T. evansi infection plays an important role in anemia. In this sense, the mechanism of anemia in trypanosomosis is greatly associated with the generation of free radicals and super oxides following lipid peroxidation. These oxidative products generally attack the cellular integrity of erythrocytes during trypanosomosis (Anosa and Kaneko, 1983; Igbokwe, 1994). Additionally, it is well known that the main adenosine deaminase (ADA) substrate, adenosine, is involved in various functions of T. evansi (Suswam et al., 2003), as well as in the pathogenesis of anemia (Zhang and Xia, 2012; Zhang et al., 2011). Therefore, considering the inflammatory response generated after T. evansi infection and its mechanism to cause anemia, the aim of this study was to assess the effects of iron supplementation on oxidative stress and on the ADA activity in rats experimentally infected by T. evansi.
uninfected group. Animals of Groups C and D were subcutaneously inoculated with 0.1 mL of blood cryopreserved, containing 4.7 × 105 of T. evansi (on Day 0). Thus, Groups A and C represented the negative and positive controls of infection (without iron treatment), respectively; while rats of Groups B and D received two doses (60 mg kg-1) of iron (Ferrodex/Agroline®, Campo Grande, Mato Grosso, Brazil), with an interval of 5 days (Sampaio, 2012), i.e., the iron was administered subcutaneously on days 1 and 6 post-infection (PI), in both groups. This protocol was approved by the Animal Welfare Committee of Universidade do Estado de Santa Catarina, under number 1.61.13.
2.2. Trypanosoma evansi isolate The cryopreserved isolate of T. evansi used in this experiment was firstly obtained from a naturally infected dog (Colpo et al., 2005). According to the literature (Tavares et al., 2011), the use of inoculum cryopreserved of T. evansi is indicate when a slow pattern of infection is required, since the cryopreservation helps to keep the parasitemia lower. This procedure prevents animal death by acute disease. Therefore, blood containing T. evansi under cryopreservation was thawed, and immediately inoculated in the animals.
2.3. Level of parasitemia The evolution of parasitemia and the effect of the treatment were daily monitored through fresh blood smears microscopically examined during 8 days PI. Each slide was mounted with blood collected from the distal region of the tail vein, stained by the panoptic method, and visualized at a magnification of 1000× (Da Silva et al., 2006). The technique requires the observation of 25 fields, and the mean parasitemia was estimated for each animal.
2.4. Sample collection Blood was collected on day 8 PI (Table 1). Part of the blood (2 mL) was stored in EDTA tubes for hemogram (1 mL), and erythrocytes separation to E-ADA analysis (1 mL). The remaining aliquot (6 mL) was stored without anticoagulant in order to obtain serum for assessment of E-ADA activity, as well as to assess the levels of iron and other biochemical variables.
2. Materials and Methods 2.5. Hemogram 2.1. Experimental design Twenty male rats (Wistar), 120-day-old, weighing an average of 420 ± 24 g were kept in cages (five rats each) throughout the experiment. They were housed in an experimental room on a light/ dark cycle of 12 h, under controlled temperature and humidity (23 ± 2 °C; 70%, respectively), fed with commercial feed and water ad libitum. All animals were submitted to a period of 10 days of adaptation before the beginning of the experiment. The animals were divided into four groups (A–D – Table 1). Groups A and B were composed of healthy rats, representing the
Total erythrocyte count, hematocrit (Ht), hemoglobin concentration (Hb), mean corpuscular volume (MCV), mean corpuscular hemoglobin concentration (MCHC), and total leukocytes were evaluated. The determination of hematocrit was performed according to the technique described by Feldman et al. (2000). Erythrocyte and leukocytes count, and hemoglobin concentration were carried out using an automatic counter (BC 2800 Vet, Mindray, Curitiba, Paraná, Brazil). Blood smears were prepared and stained according to Romanowski’s method, allowing the evaluation of cell morphology and leukocytes differentiation.
N.B. Bottari et al./Experimental Parasitology 147 (2014) 1–6
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Table 1 Experimental design of rats infected by Trypanosoma evansi and subjected to iron supplementation: groups, inoculation day, doses of iron administered and sampling time. Groups
Infected or uninfected
Inoculation
Two doses of irona
Sampling
A B C D
Uninfected Uninfected Infected by T. evansi Infected by T. evansi
– – Day 0 Day 0
– Days 1 and 6 PI – Days 1 and 6 PI
Day 8 PI Day 8 PI Day 8 PI Day 8 PI
Iron was administered subcutaneously on days 1 and 6 post-infection (PI).
2.6. Biochemical variables 2.6.1. ADA activity in erythrocytes and serum Firstly, erythrocytes were obtained from whole blood preserved with EDTA according to the technique described by Hostetter and Johnson (1989). Erythrocytes were resuspended in Hepes buffer at 10% in order to assess the hematocrit. The blood was centrifuged at 3500× g for 10 min, allowing serum separation. All samples were frozen (−20 °C) until analysis. Then, ADA activity was measured spectrophotometrically in the erythrocytes and serum by the method described by Giusti and Gakis (1971). The reaction was started by addition of the substrate (adenosine) to a final concentration of 21 mmol/L and incubations were carried out for 1 h at 37 °C. The reaction was stopped by adding 106 mmol/L/0.16 mmol/L phenolnitroprusside/mL solution. The reaction mixtures were immediately mixed to 125 mmol/L/11 mmol/L alkalinehypochlorite (sodium hypochlorite) and vortexed. Ammonium sulphate at a concentration of 75 μmol/L was used as ammonium standard. The ammonia concentration is directly proportional to the absorption of indophenol at 650 nm. The specific activity is reported as U/g of Hb in erythrocytes and U/L in serum. 2.6.2. Seric iron levels The quantification of iron in serum was performed using a commercial kit (Biotécnica Indústria & Comércio Ltda, Varginha, Minas Gerais, Brazil) on a semi-automatic analyzer Bio-2000 (BioPlus Ltda, Barueri, São Paulo, Brazil) according to manufacturer recommendations. The results were expressed in μg dL−1. 2.6.3. Serum activity of alanine aminotransferase (ALT), creatinine (Cr), total protein (tp) and urea (U) ALT activity, levels of Cr, TP and U were evaluated in a semiautomatic analyzer (BioPlus Ltda ® , São Paulo, Brazil) using commercial kits (Analiza®, Gold Analisa Diagnóstica Ltda, Belo Horizonte, Minas Gerais, Brazil). Tests were carried out in duplicate. 2.6.4. Advanced oxidation protein products (AOPP), total oxidant status (TOS), ferric reducing ability of plasma (FRAP), total antioxidant capacity (TAC) and nitrite/nitrate levels (NOx) AOPP, TOS, FRAP, TAC and NOx were evaluated in order to identify the oxidative status. These measurements were performed on Cobas Mira® (Cobas Mira®, Canoas, Rio Grande do Sul, Brazil) automated system according to standardized techniques. Serum AOPP levels were evaluated according to the method described by Hanasand et al. (2012), and results were expressed as μmol L−1. Serum TOS levels were determined using a novel automated measurement method developed by Erel (2005), in which the oxidant present in the sample oxidize the ferrous ion-o-dianisidine complex to ferric ion. The results were expressed as μmol H2O2 Equivalent L−1. Serum TAC levels were assessed thorough a novel automated method (Erel, 2004), involving the production of hydroxyl radical. The antioxidant effect of the sample against the potent free radical reactions initiated by the hydroxyl radicals produced was measured, and the results were expressed as mmol Trolox-equivalents L−1. The total antioxidant potential on serum samples was determined by the FRAP
assay of Benzie and Strain (1996). FRAP assay measures the change in the absorbance at 593 nm after the formation of a blue colored Fe2+ tripyridyltriazine compound from colorless oxidized Fe3+ form, by the action of electron donation to antioxidants. Results were expressed as μmol/L. Finally, serum nitric oxide (NO) levels were analyzed indirectly, quantifying seric NOx according to the technique described by Tatsch et al. (2011) with results expressed as μmol L−1.
2.7. Statistical analysis Initially, normality test was applied to verify if the data had a normal distribution. Leucogram data did not show normal distribution and, thus, they were transformed to logarithmic form. Then, all data of this study were submitted to analysis of variance (oneway ANOVA) followed by Duncan test (P < 0.05). 3. Results 3.1. Disease course We did not observe differences in pre-patent period in group D when compared with group C throughout this study; however it was observed a decreased number of trypomastigotes form in the blood of animals from group D when compared with group C on day 8 PI (P < 0.05). Data are shown in Fig. 1. 3.2. Hematological parameters Full data of hematological parameters are shown in Table 2. Animals infected by T. evansi (Groups C and D) showed a decrease in the erythrocyte count, hematocrit and hemoglobin when compared with uninfected animals. These same differences were not observed in groups A and B (P > 0.05). Animals infected by T. evansi showed an increase in the number of total leukocytes, lymphocytes, and monocytes when compared with uninfected animals (P < 0.05).
Trypanosomes/field (1000x)
a
12 Group C
10 8
Group D
6 4 2 0 1
2
3
4
5
6
7
8
Days post-infection Fig. 1. Average parasitemia of animals infected with Trypanosoma evansi corresponding to Groups C and D (infected). Note: Group D was supplemented with iron (0.6 mL kg−1, subcutaneously).
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Table 2 Mean and standard deviation of haematological variables after iron supplementation on rats experimentally infected by Trypanosoma evansi. Variables
Uninfected
6
−1
Total erythrocytes (×10 μL ) Hematocrit (%) Hemoglobin (g dL−1) MCV (fl) MCHC (%) Total Leukocytes (×103 μL−1) Neutrophils (×103 μL−1) Lymphocytes (×103 μL−1) Monocytes (×103 μL−1) Eosinophils (×103 μL−1)
Infected
Group A (untreated)
Group B (treated)
Group C (untreated)
Group D (treated)
8.1 ± 0.31 43.8 ± 1.6a 13.0 ± 0.47a 54.1 ± 0.4a 30.3 ± 0.5a 5.6 ± 1.3b 1.4 ± 0.8a 4.0 ± 0.9b 0.015 ± 0.034b 0.10 ± 0.12a
8.2 ± 0.27 46.6 ± 1.9a 13.7 ± 0.40a 55.4 ± 2.1ab 30.1 ± 0.4a 6.4 ± 1.9b 2.1 ± 0.9a 4.1 ± 1.1b 0.023 ± 0.053b 0.22 ± 0.31a
6.3 ± 0.41 33.3 ± 3.1c 10.5 ± 1.0c 52.4 ± 1.0b 31.1 ± 0.7a 11.1 ± 3.9a 1.5 ± 0.8a 9.0 ± 3.0a 0.46 ± 0.18a 0.075 ± 0.044a
7.5 ± 0.32b 39.9 ± 3.6b 11.8 ± 0.8b 53.6 ± 1.6ab 31.2 ± 0.8a 10.1 ± 3.1a 1.7 ± 1.3a 7.8 ± 2.4a 0.43 ± 0.28a 0.14 ± 0.07a
a
a
c
Means followed by the same letter in the same line do not differ significantly by the Duncan test (P > 0.05).
uninfected groups (P > 0.05). Total proteins, urea, and creatinine did not differ among groups (P > 0.05).
3.3. Biochemical parameters Full data of biochemical variables, oxidative and antioxidants markers are shown in Table 3. 3.3.1. ADA activity in serum and erythrocytes A decrease on seric ADA activity in animals from group C was observed when compared with uninfected animals (P < 0.05). The iron treatment did not cause significant changes on the activity of ADA in serum of rats from group D when compared with the negative group (P > 0.05). In erythrocytes, a decreased (P < 0.05) activity of E-ADA on group C was observed when compared with the uninfected; however animals from group D showed an increase on ADA activity (P < 0.05), when compared with groups A and group C, both groups that did not receive iron supplementation. 3.3.2. Iron levels No differences on seric iron levels were observed between uninfected groups (Group A and B – P > 0.05), while animals from group C showed decreased iron levels in serum when compared with uninfected rats (P < 0.05); however, animals from group D maintained a similar level of seric iron, as the negative control groups (groups A and B). 3.3.3. ALT No difference was observed on seric levels of ALT between uninfected groups (P > 0.05), but, in group C, an increase on seric levels of ALT (P < 0.05) was observed. In group D, the animals did not show changes in seric levels of ALT when compared with the
3.3.4. AOPP, TOS, FRAP, TAC and NOx Rats from group B showed an increase in AOPP levels compared with uninfected animals (P < 0.05 – Group A). A similar result was observed on groups C and D. Additionally, animals of group D presented decreased TOS levels when compared with other groups (P < 0.05). The negative (A) and positive (C) control groups did not show differences on NOx levels (P < 0.05). However, the opposite situations were observed in animals from groups B and D (supplemented with iron), where decreased levels of NOx were observed, when compared with the other groups (P < 0.05). No difference was observed between uninfected groups on seric FRAP levels (P > 0.05); but animals of group D showed increased FRAP levels when compared with the other groups (P < 0.05). TAC levels did not differ among groups (P > 0.05). 4. Discussion In the present study, rats infected by T. evansi showed reduced values for erythrocytes, hematocrit, and hemoglobin as reported by other authors (França et al., 2011; Menezes et al., 2004; Wolkmer et al., 2009), corroborating with the fact that anemia is a common feature of T. evansi infections (Da Silva et al., 2009; Herrera et al., 2004). Therefore, the reduction of hematological parameters might be a direct consequence of parasitemia, since the parasitemia causes a large number of red blood cells (RBCs) to be removed from circulation by the mononuclear phagocytic system (MPS) in the spleen, bone marrow, and lymph nodes. The removal of a large number of
Table 3 Mean and standard deviation of biochemical variables after iron supplementation on rats experimentally infected by Trypanosoma evansi. Variables
ADA in serum (U L−1) ADA in erythrocytes (U g−1 of Hb) Iron (mg L−1) Total protein (mg dL−1) ALT (U L−1) Urea (mg dL−1) Creatinine (mg dL−1) AOPP (μmol L−1) TOS (μmol H2O2 Equivalent L−1) TAC (×103 mmol Trolox Equivalent L−1) FRAP (×102 μmol L−1) NOx (μmol L−1)
Uninfected
Infected
Group A (untreated)
Group B (treated)
Group C (untreated)
Group D (treated)
6.1 ± 2.0a 0.56 ± 0.07b 3.14 ± 0.21a 8.36 ± 0.09a 52.2 ± 3.1b 43.3 ± 4.7a 0.80 ± 0.09a 38.3 ± 1.0b 23.5 ± 5.9b 1.11 ± 0.05a 5.83 ± 0.77b 104.7 ± 25b
5.8 ± 1.9a 0.64 ± 0.20ab 3.02 ± 0.34a 8.50 ± 0.3a 49.3 ± 3.9b 40.2 ± 5.6a 0.74 ± 0.03a 42.5 ± 0.9a 19.9 ± 5.1b 1.08 ± 0.09a 4.81 ± 1.12b 54.9 ± 27c
3.3 ± 0.8b 0.32 ± 0.1c 1.25 ± 0.18b 8.3 ± 0.5a 64.2 ± 5.1a 44.0 ± 3.4a 0.69 ± 0.04a 43.8 ± 2.1a 25.8 ± 7.2b 1.04 ± 0.12a 5.93 ± 1.5b 128.6 ± 18a
4.8 ± 1.5ab 0.96 ± 0.31a 2.45 ± 0.22ab 8.0 ± 0.4a 49.2 ± 7.3b 39.6 ± 7.8a 075 ± 0.06a 44.8 ± 2.3a 3.4 ± 5.6a 1.04 ± 0.08a 7.69 ± 1.9a 63.4 ± 14c
Note: Animals on groups A and B were not infected, while animals on groups C and D were intraperitoneally infected with trypomastigotes forms of Trypanosoma evansi. Means followed by the same letter in the same line do not differ significantly by the Duncan test (P > 0.05).
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RBCs leads to a drop in packed red cell volume (PCV) to below 25%, reaching to as low as 10% (Matios and Eyob, 2013). It is possible to correlate our hematological findings with the fact that oxidative damage to red blood cells has been related to the generation of free radicals (Vives-Corrons et al., 1995), and that ironreplacement treatment decreases antioxidant stress by regulating enzymatic levels (Isler et al., 2002), which shows that our results are relevant in T. evansi infection. In the current study, iron supplementation led to an increase in AOPP, but it was not able to increase the levels of TOS. Iron is required for the activity of numerous ironand heme-containing proteins, but “free” (i.e. redox active) iron catalyzes the formation of highly toxic reactive oxygen species (ROS) (Graf et al., 1984) that damage lipids, proteins, and DNA (Quinlan et al., 2002), facts that may explain the elevation on AOPP levels. On the other hand, animals infected and treated with iron showed increased FRAP levels, demonstrating additionally the antioxidant strength of iron by reducing Fe3+ to Fe2+ (Benzie and Strain, 1996). The formation of highly toxic ROS due to the iron supplementation, even though is not beneficial to the host, may collaborate to moderate the parasitemia, since the infected animals that were supplemented with iron presented lower parasitemia that the ones infected not supplemented. According to Jain (1986), there are two phases of anemia, which is the most prominent finding in natural trypanosome infections, and the severity of the first phase is proportional to the degree of parasitemia. Thus, iron supplementation may indirectly collaborate to enhance the host response against T. evansi by diminishing the parasitemia. Iron supplementation had also direct effect on NOx levels, since this metal plays an inhibitory role on the expression of inducible NOx synthase (iNOS), which is an inflammatory setting, often accompanied by a large production of ROS gene (Vallabhapurapu and Karin, 2009). This inhibitory action might have collaborated to the reduced levels of NOx observed in the groups supplemented with iron, and also helped to explain the increased levels of ALT in rats infected and not supplemented with iron. Rats infected by T. evansi showed increase level of ALT (Barr, 1991; Monzón and Villavicencio, 1990), but many other studies also demonstrated that hepatocytes express iNOS following exposure to various cytokines such as tumor necrosis factor (TNF), interleukin 1(IL1), interferon gamma (IFNγ), and interleukin six (IL-6) (Adamson and Billings, 1993; Curran et al., 1990; Geller et al., 1993), a pro-inflammatory condition already reported on infections by T. evansi (Paim et al., 2011). Therefore, the presence of free iron into the bloodstream may help to inhibit iNOS, modulating the pro-inflammatory response against the parasite and, also, the concomitant cellular damage to cells and tissues. Similarly to our study, the reduction of ADA activity in serum and erythrocytes of rats infected with T. evansi has been reported previously (Da Silva et al., 2011). Regarding the ADA activity, it is possible to correlate this enzyme directly with the major effect of this parasitic disease, the anemia. It is well known that the main ADA substrate, adenosine, is involved in various functions of T. evansi (Suswam et al., 2003), as well as in the pathogenesis of anemia (Zhang and Xia, 2012; Zhang et al., 2011). In this sense, early in 1990, researchers reported a relationship between iron deficiency and anemia, concluding that iron depletion impairs the ADA activity (Luo et al., 1990). The same authors reported that iron supplementation was able to increase ADA activity in anemic animals, correlating the changes in the enzymatic activity with the iron deficiency. Our results did not show that levels of ADA statistically increased in groups supplemented with iron; however, they showed that the group infected and not supplemented presented reduced activity of ADA (along with reduction in hematologic parameters) in serum and erythrocytes, corroborating with reports of Luo and collaborators (Luo et al., 1990). Based on these results, we can conclude that the iron supplementation was able to provide some effect on the oxidative stress
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imbalance, as well as in the ADA activity modulation. Iron supplementation helped to diminish the parasitemia, and indirectly reduced the establishment of the anemic process; and it was capable of modulating the activity of ADA in serum and erythrocytes, maintaining its levels as equal as in the uninfected groups. Iron supplementation was also able to inhibit iNOS, reducing NOx levels and preventing increase of ALT. Meanwhile, FRAP levels were reduced modulating an oxidative stress situation. However, it was also possible to observe that free iron generated an increase in AOPP levels probably due to protein and DNA damage caused by the association between parasite action and free iron bioavailability.
References Adamson, G.M., Billings, R.E., 1993. Cytokine toxicity and induction of NO sythase activity in cultured mouse hepatocytes. Toxicol. Appl. Pharmacol. 119, 100–107. Anosa, V.O., Kaneko, J.J., 1983. Pathogenesis of Trypanosoma brucei infection in deer mice (Peromyscus maniculatus): hematologic, erythrocyte biochemical, and ironmetabolic aspects. Am. J. Vet. Res. 44, 639–644. Barr, S.C., 1991. American trypanosomiasis in dog. Comp. Cont. Educ. Pract. Vet. 13, 745–752. Benzie, I.F.F., Strain, J.J., 1996. The ferric reducing ability of plasma (FRAP) as a measure of “Antioxidant Power”: The FRAP Assay. Anal. Biochem. 239, 70–76. Calder, P.C., Albers, R., Antoine, J.M., Blum, S., Bourdet-Sicard, R., Ferns, G.A., et al., 2009. Inflammatory disease processes and interactions with nutrition. Br. J. Nutr. 101, 1–45. Colpo, C.B., Monteiro, S.G., Stainki, D.R., 2005. Natural infection by Trypanosoma evansi in dogs. Ciênc. Rural 35, 717–719. Curran, R.D., Billiar, T.R., Stuer, D.J., Ochoa, J.B., Harbrecht, B.G., Flint, S.G., 1990. Multiple cytokines are required to induce hepatocyte nitric oxide production and inhibit total protein synthesis. Ann. Surg. 212, 462–469. Da Silva, A.S., Doyle, R.L., Monteiro, S.G., 2006. Métodos de contenção e confecção de esfregaço sanguíneo para pesquisa de hemoparasitas em ratos e camundongos. FZVA. 13, 83–87. Da Silva, A.S., Costa, M.M., Wolkmer, P., Zanette, R.A., Faccio, L., Gressler, L.T., et al., 2009. Trypanosoma evansi: hematologic changes in experimentally infected cats. Exp. Parasitol. 123, 31–34. Da Silva, A.S., Bellé, L.P., Bitencourt, P.E.R., Souza, V.C.G., Costa, M.M., Oliveira, C.B., et al., 2011. Activity of the enzyme adenosine deaminase in serum, erythrocytes and lymphocytes of rats infected with Trypanosoma evansi. Parasitology 138, 201–208. Doherty, C.P., 2007. Host-pathogen interactions: the role of iron. J. Nutr. 137, 1341–1344. Erel, O., 2004. A novel automated method to measure total antioxidant response against potent free radical reactions. Clin. Biochem. 37, 112–119. Erel, O., 2005. A new automated colorimetric method for measuring total oxidant status. Clin. Biochem. 38, 1103–1111. Feldman, B.F., Zinkl, J.G.A., Jain, N.C., 2000. Schalm’s Veterinary Hematology, fifth edition. Lippincott Williams & Wilkins, Philadelphia, PA, p. 787. França, R.T., Da Silva, A.S., Wolkmer, P., Oliveira, V.A., Pereira, M.E., Leal, M.L.R., et al., 2011. Delta-aminolevulinate dehydratase activity in red blood cells of rats infected with Trypanosoma evansi. Parasitology 138, 1272–1277. Geller, D.A., Nussler, A.K., Di Silvio, M., 1993. Cytokines, endotoxin and glucocorticoids regulate the expression of inducible nitric oxide synthase in hepatocytes. PNAS 90, 522–526. Giusti, G., Gakis, C., 1971. Temperature conversion factors, activation energy, relative substrate specificity and optimum pH of adenosine deaminase from human serum and tissues. Enzyme 12, 417–420. Graf, E., Mahoney, J.R., Bryant, R.G., Eaton, J.W., 1984. Iron-catalyzed hydroxyl radical formation. Stringent requirement for free iron coordination site. J. Biol. Chem. 259, 3620–3624. Hanasand, M., Omdal, R., Norheim, K.B., Goransson, L.G., Brede, C., Jonsson, G., 2012. Improved detection of advanced oxidation protein products in plasma. Clin. Chim. Acta 413, 901–906. Herrera, H.M., Dávila, A.M., Norek, A., Abreu, U.G., Souza, S.S., 2004. Enzootiology of Trypanosoma evansi in Pantanal, Brazil. Vet. Parasitol. 11, 366–369. Hostetter, M.K., Johnson, G.M., 1989. The erythrocyte as investigator of inflammation. Generation of amidated C3 by erythrocyte adenosine deaminase. J. Clin. Invest. 84, 665–671. Iannotti, L.L., Tielsch, J.M., Black, M.M., Black, R.E., 2006. Iron supplementation in early childhood: health benefits and risks. Am. J. Clin. Nutr. 84, 1261–1276. Igbokwe, I.O., 1994. Increased susceptibility of erythrocytes to in vitro peroxidation in acute Trypanosoma brucei infection of mice. Vet. Parasitol. 55, 279–286. Isler, M., Delibas, N., Guclu, M., Gultekin, F., Sutcu, R., Bahceci, M., et al., 2002. Superoxide dismutase and glutathione peroxidase in erythrocytes of patients with iron deficiency anemia: effects of different treatment modalities. Croat. Med. J. 43, 16–19. Jain, N.C., 1986. Schalm’s Veterinary Hematology, fourth edition. Lea & Febiger, Philadelphia, PA. 1221p.
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N.B. Bottari et al./Experimental Parasitology 147 (2014) 1–6
Luo, C., Chen, J., Liao, Q., Li, Q., Yang, X., Li, Y., 1990. Influence of iron deficiency anemia on development of thymus and spleen and adenosine deaminase activity in rats. Hua Xi Yi Ke Da Xue Xue Bao 21, 63–66. Matios, L., Eyob, E., 2013. Review on camel trypanosomosis (surra) due to Trypanosoma evansi: Epidemiology and host response. J. Vet. Med. Anim. Health 5, 334–343. Menezes, V.T., Queiroz, A.O., Gomes, M.A.M., Marques, M.A.P., Jansen, A.M., 2004. Trypanosoma evansi in inbred and Swiss-Webster mice: distinct aspects of pathogenesis. Parasitol. Res. 94, 193–200. Monzón, C.M., Villavicencio, V.I., 1990. Serum protins inguinea-pigs and horses infected with Trypanosoma evansi (Steel, 1885). Vet. Parasitol. 36, 295–301. Omer, O.H., Mousa, H.M., Al-Wabel, N., 2007. Study on the antioxidant status of rats experimentally infected with Trypanosoma evansi. Vet. Parasitol. 145, 142–145. Paim, F.C., Duarte, M.M., Costa, M.M., Da Silva, A.S., Wolkmer, P., Silva, C.B., et al., 2011. Cytokines in rats experimentally infected with Trypanosoma evansi. Exp. Parasitol. 128, 365–370. Quinlan, G.J., Evans, T.W., Gutteridge, J.M., 2002. Iron and the redox status of the lungs. Free Radic. Biol. Med. 33, 1306–1313. Rodrigues, A., Fighera, R.A., Souza, T.M., Schild, A.L., Soares, M.P., 2005. Surtos de tripanossomíase por Trypanosoma evansi em equinos no Rio Grande do Sul: aspectos epidemiológicos, clínicos, hematológicos e patológicos. Pesqui. Vet. Bras. 25, 239–249. Sampaio, A.F.S., 2012. Effects of Supplementation with Injectable Iron on the Production of ROS by Granulocytes and Oxidative Damage in Cardiac and Pancreatic Tissues of Diabetic Rats (Dissertation, Master in Health and Nutrition). Universidade Federal de Ouro Preto, Ouro Preto, Brazil. 96p. Silva, R.A.M.S., Seidl, A., Ramirez, L., Dávila, A.M.R., 2002. Trypanosoma evansi e Trypanosoma vivax: Biologia, Diagnóstico e controle. Corumbá, Embrapa Pantanal.
Smith, A., 2002. Homeostasis of heme in health and disease: current aspects of the structural biology of heme-protein interactions and of gene regulation. DNA Cell Biol. 21, 245–249. Suswam, E.A., Ross, C.A., Martin, R.J., 2003. Changes in adenosine transport associated with melaminophenyl arsenical (Mel CY) resistance in Trypanosoma evansi: down-regulation and affinity changes of the P2 transporter. Parasitology 127, 543–549. Tatsch, E., Bochi, G.V., Pereira, R.S., Kober, H., Agertt, V.A., De Campos, M.M.A., et al., 2011. A simple and inexpensive automated technique for measurement of serum nitrite/nitrate. Clin. Biochem. 44, 348–350. Tavares, K.C.S., Da Silva, A.K., Wolkmer, P., Monteiro, S.G., Miletti, L.C., 2011. Cryopreservation of Trypanosoma evansi after DEAE-cellulose purificarion: Evaluation of infective parameters. Res. Vet. Sci. 90, 257–259. Vallabhapurapu, S., Karin, M., 2009. Regulation and function of NF-kappaB transcription factors in the immune system. Annu. Rev. Immunol. 27, 693–733. Vives-Corrons, J.L., Miguel-Garcia, A., Pujades, M.A., Miguel-Sosa, A., Cambiazzo, S., Linares, M., et al., 1995. Increased susceptibility of microcytic red blood cells to in vitro oxidative stress. Eur. J. Haematol. 55, 327–331. Weinberg, E.D., 1978. Iron and infection. Microbiol. Rev. 42, 45–66. Wolkmer, P., Silva, A.S., Cargnelutti, J., Costa, M.M., Traesel, C.K., Lopes, S.T.A., et al., 2007. Resposta eritropoética de ratos em diferentes graus de parasitemia por Trypanosoma evansi. Ciênc. Rural 37, 1682–1687. Wolkmer, P., da Silva, A.S., Traesel, C.K., Paim, F.C., Cargnelutti, J.F., Pagnoncelli, M., et al., 2009. Lipid peroxidation associated with anemia in rats experimentally infected with Trypanosoma evansi. Vet. Parasitol. 165, 41–46. Zhang, Y., Xia, Y., 2012. Adenosine signaling in normal and sickle erythrocytes and beyond. Microbes Infect. 14, 863–873. Zhang, Y., Dai, Y., Wen, J., Zhang, W., Grenz, A., Sun, H., et al., 2011. Detrimental effects of adenosine signaling in sickle cell disease. Nat. Med. 17, 79–86.
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Experimental Parasitology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / y e x p r
Full length article
Effects of iron supplementation on blood adenine deaminase activity and oxidative stress in Trypanosoma evansi infection of rats Nathieli B. Bottari a,b, Matheus D. Baldissera a, Alexandre A. Tonin a, Raqueli T. França c, Danieli Zanini b, Marta L.R. Leal d, Sonia T.A. Lopes c, Maria Rosa C. Schetinger b, Vera M. Morsch b, Silvia G. Monteiro a, Naiara S. Guarda f, Rafael N. Moresco f, Adelina R. Aires d, Lenita M. Stefani e, Aleksandro S. Da Silva b,e,* a
Department of Microbiology and Parasitology, Universidade Federal de Santa Maria (UFSM), Santa Maria, RS, Brazil Toxicological Biochemistry of Graduate Program, UFSM, Santa Maria, RS, Brazil c Department of Small Animal, UFSM, Santa Maria, RS, Brazil d Department of Large Animal, UFSM, Santa Maria, RS, Brazil e Department of Animal Science, Universidade do Estado de Santa Catarina (UDESC), Chapecó, SC, Brazil f Department of Clinical and Toxicological Analysis, UFSM, Santa Maria, RS, Brazil b
H I G H L I G H T
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G R A P H I C A L
Trypanosoma evansi is the etiological agent of the disease known as “Surra” or “Mal das cadeiras”. T. evansi infection plays an important role in the anemia, iron levels, ADA activity and oxidative stress. Iron supplementation was able to provide some effect on the oxidative stress imbalance, as well as in the ADA modulation. Iron supplementation helped to diminish the parasitemia, and indirectly reduced the anemic process.
A R T I C L E
I N F O
Article history: Received 16 April 2014 Received in revised form 11 July 2014 Accepted 15 September 2014 Available online 6 October 2014 Keywords: Trypanosomosis Anemia ADA Oxidants Antioxidants
Infected by Trypanosoma evansi – day 0
↗
and Iron supplementation at days 1 and 6 postinfection
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Supplementation was able to provide some effect on the oxidative stress imbalance and anemia, as well as in the ADA activity modulation
A B S T R A C T
The aim of this study was to assess the effects of iron supplementation on oxidative stress and on the activity of the adenosine deaminase (ADA) in rats experimentally infected by Trypanosoma evansi. For this purpose, 20 rats were divided into four experimental groups with five animals each as follows: groups A and B were composed by healthy animals, while animals from groups C and D were infected by T. evansi. Additionally, groups B and D received two subcutaneous doses of iron (60 mg kg-1) within an interval of 5 days. Blood samples were drawn on day 8 post infection in order to assess hematological and biochemical variables. Among the main results are: (1) animals from group C showed reduced erythrogram (with tendency to anemia); however the same results were not observed for group D; this might be a direct effect of free iron on trypanosomes which helped to reduce the parasitemia and the damage to erythrocytes caused by the infection; (2) iron supplementation was able to reduce NOx levels by inhibiting iNOS, and thus, providing an antioxidant action and, indirectly, reducing the ALT levels in groups B
* Corresponding author. Fax: +55 49 3330-9440. E-mail address: [email protected] (A.S. Da Silva). http://dx.doi.org/10.1016/j.exppara.2014.09.002 0014-4894/© 2014 Elsevier Inc. All rights reserved.
A B S T R A C T
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and D; (3) increase FRAP levels in group D; (4) reduce ADA activity in serum and erythrocytes in group C; however, this supplementation (5) increased the protein oxidation in groups B and D, as well as group C (positive control). Therefore, iron showed antioxidant and oxidant effects on animals that received supplementation; and it maintained the activity of E-ADA stable in infected/supplemented animals. © 2014 Elsevier Inc. All rights reserved.
1. Introduction Trypanosoma evansi is the etiological agent of the disease known as “Surra” or “Mal das cadeiras” in horses. It shows a wide geographical distribution, being found parasitizing various species of domestic and wild animals (Silva et al., 2002). Rapid weight loss, anemia, swollen limbs, progressive weakness, neurological signs and paralysis of the hind limbs (in chronic cases) are the clinical signs usually observed in trypanosomosis (Herrera et al., 2004; Rodrigues et al., 2005). The disease in its acute form is characterized by hematological changes, progressive anemia, anorexia, leading in some cases to death (Omer et al., 2007). T. evansi infection is able to cause an inflammatory response in the host (Paim et al., 2011). Considering that inflammation is a stereotypical physiological response to infections and tissue injury, which leads to pathogen killing as well as tissue repair processes, helping to restore homeostasis at infected or damaged sites (Calder et al., 2009), it is important to consider on this conditions, may changes in the plasma level of certain trace metals (e.g., a reduction in plasma iron) can be observed (Weinberg, 1978). Iron is an essential nutrient for biochemical processes, such as electron transfer and regulation of cell growth, transport oxygen. It is also essential for cellular homeostasis, DNA synthesis, energetic metabolism and synthesis of heme group (Iannotti et al., 2006; Smith, 2002). Iron redox enzymes are essential for cell respiration, DNA synthesis, and free radical-scavenging mechanisms, but in the presence of an inflammatory process, we observe a lack of free iron (Doherty, 2007). According to Wolkmer et al. (2007) and Da Silva et al. (2009) T. evansi infection plays an important role in anemia. In this sense, the mechanism of anemia in trypanosomosis is greatly associated with the generation of free radicals and super oxides following lipid peroxidation. These oxidative products generally attack the cellular integrity of erythrocytes during trypanosomosis (Anosa and Kaneko, 1983; Igbokwe, 1994). Additionally, it is well known that the main adenosine deaminase (ADA) substrate, adenosine, is involved in various functions of T. evansi (Suswam et al., 2003), as well as in the pathogenesis of anemia (Zhang and Xia, 2012; Zhang et al., 2011). Therefore, considering the inflammatory response generated after T. evansi infection and its mechanism to cause anemia, the aim of this study was to assess the effects of iron supplementation on oxidative stress and on the ADA activity in rats experimentally infected by T. evansi.
uninfected group. Animals of Groups C and D were subcutaneously inoculated with 0.1 mL of blood cryopreserved, containing 4.7 × 105 of T. evansi (on Day 0). Thus, Groups A and C represented the negative and positive controls of infection (without iron treatment), respectively; while rats of Groups B and D received two doses (60 mg kg-1) of iron (Ferrodex/Agroline®, Campo Grande, Mato Grosso, Brazil), with an interval of 5 days (Sampaio, 2012), i.e., the iron was administered subcutaneously on days 1 and 6 post-infection (PI), in both groups. This protocol was approved by the Animal Welfare Committee of Universidade do Estado de Santa Catarina, under number 1.61.13.
2.2. Trypanosoma evansi isolate The cryopreserved isolate of T. evansi used in this experiment was firstly obtained from a naturally infected dog (Colpo et al., 2005). According to the literature (Tavares et al., 2011), the use of inoculum cryopreserved of T. evansi is indicate when a slow pattern of infection is required, since the cryopreservation helps to keep the parasitemia lower. This procedure prevents animal death by acute disease. Therefore, blood containing T. evansi under cryopreservation was thawed, and immediately inoculated in the animals.
2.3. Level of parasitemia The evolution of parasitemia and the effect of the treatment were daily monitored through fresh blood smears microscopically examined during 8 days PI. Each slide was mounted with blood collected from the distal region of the tail vein, stained by the panoptic method, and visualized at a magnification of 1000× (Da Silva et al., 2006). The technique requires the observation of 25 fields, and the mean parasitemia was estimated for each animal.
2.4. Sample collection Blood was collected on day 8 PI (Table 1). Part of the blood (2 mL) was stored in EDTA tubes for hemogram (1 mL), and erythrocytes separation to E-ADA analysis (1 mL). The remaining aliquot (6 mL) was stored without anticoagulant in order to obtain serum for assessment of E-ADA activity, as well as to assess the levels of iron and other biochemical variables.
2. Materials and Methods 2.5. Hemogram 2.1. Experimental design Twenty male rats (Wistar), 120-day-old, weighing an average of 420 ± 24 g were kept in cages (five rats each) throughout the experiment. They were housed in an experimental room on a light/ dark cycle of 12 h, under controlled temperature and humidity (23 ± 2 °C; 70%, respectively), fed with commercial feed and water ad libitum. All animals were submitted to a period of 10 days of adaptation before the beginning of the experiment. The animals were divided into four groups (A–D – Table 1). Groups A and B were composed of healthy rats, representing the
Total erythrocyte count, hematocrit (Ht), hemoglobin concentration (Hb), mean corpuscular volume (MCV), mean corpuscular hemoglobin concentration (MCHC), and total leukocytes were evaluated. The determination of hematocrit was performed according to the technique described by Feldman et al. (2000). Erythrocyte and leukocytes count, and hemoglobin concentration were carried out using an automatic counter (BC 2800 Vet, Mindray, Curitiba, Paraná, Brazil). Blood smears were prepared and stained according to Romanowski’s method, allowing the evaluation of cell morphology and leukocytes differentiation.
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Table 1 Experimental design of rats infected by Trypanosoma evansi and subjected to iron supplementation: groups, inoculation day, doses of iron administered and sampling time. Groups
Infected or uninfected
Inoculation
Two doses of irona
Sampling
A B C D
Uninfected Uninfected Infected by T. evansi Infected by T. evansi
– – Day 0 Day 0
– Days 1 and 6 PI – Days 1 and 6 PI
Day 8 PI Day 8 PI Day 8 PI Day 8 PI
Iron was administered subcutaneously on days 1 and 6 post-infection (PI).
2.6. Biochemical variables 2.6.1. ADA activity in erythrocytes and serum Firstly, erythrocytes were obtained from whole blood preserved with EDTA according to the technique described by Hostetter and Johnson (1989). Erythrocytes were resuspended in Hepes buffer at 10% in order to assess the hematocrit. The blood was centrifuged at 3500× g for 10 min, allowing serum separation. All samples were frozen (−20 °C) until analysis. Then, ADA activity was measured spectrophotometrically in the erythrocytes and serum by the method described by Giusti and Gakis (1971). The reaction was started by addition of the substrate (adenosine) to a final concentration of 21 mmol/L and incubations were carried out for 1 h at 37 °C. The reaction was stopped by adding 106 mmol/L/0.16 mmol/L phenolnitroprusside/mL solution. The reaction mixtures were immediately mixed to 125 mmol/L/11 mmol/L alkalinehypochlorite (sodium hypochlorite) and vortexed. Ammonium sulphate at a concentration of 75 μmol/L was used as ammonium standard. The ammonia concentration is directly proportional to the absorption of indophenol at 650 nm. The specific activity is reported as U/g of Hb in erythrocytes and U/L in serum. 2.6.2. Seric iron levels The quantification of iron in serum was performed using a commercial kit (Biotécnica Indústria & Comércio Ltda, Varginha, Minas Gerais, Brazil) on a semi-automatic analyzer Bio-2000 (BioPlus Ltda, Barueri, São Paulo, Brazil) according to manufacturer recommendations. The results were expressed in μg dL−1. 2.6.3. Serum activity of alanine aminotransferase (ALT), creatinine (Cr), total protein (tp) and urea (U) ALT activity, levels of Cr, TP and U were evaluated in a semiautomatic analyzer (BioPlus Ltda ® , São Paulo, Brazil) using commercial kits (Analiza®, Gold Analisa Diagnóstica Ltda, Belo Horizonte, Minas Gerais, Brazil). Tests were carried out in duplicate. 2.6.4. Advanced oxidation protein products (AOPP), total oxidant status (TOS), ferric reducing ability of plasma (FRAP), total antioxidant capacity (TAC) and nitrite/nitrate levels (NOx) AOPP, TOS, FRAP, TAC and NOx were evaluated in order to identify the oxidative status. These measurements were performed on Cobas Mira® (Cobas Mira®, Canoas, Rio Grande do Sul, Brazil) automated system according to standardized techniques. Serum AOPP levels were evaluated according to the method described by Hanasand et al. (2012), and results were expressed as μmol L−1. Serum TOS levels were determined using a novel automated measurement method developed by Erel (2005), in which the oxidant present in the sample oxidize the ferrous ion-o-dianisidine complex to ferric ion. The results were expressed as μmol H2O2 Equivalent L−1. Serum TAC levels were assessed thorough a novel automated method (Erel, 2004), involving the production of hydroxyl radical. The antioxidant effect of the sample against the potent free radical reactions initiated by the hydroxyl radicals produced was measured, and the results were expressed as mmol Trolox-equivalents L−1. The total antioxidant potential on serum samples was determined by the FRAP
assay of Benzie and Strain (1996). FRAP assay measures the change in the absorbance at 593 nm after the formation of a blue colored Fe2+ tripyridyltriazine compound from colorless oxidized Fe3+ form, by the action of electron donation to antioxidants. Results were expressed as μmol/L. Finally, serum nitric oxide (NO) levels were analyzed indirectly, quantifying seric NOx according to the technique described by Tatsch et al. (2011) with results expressed as μmol L−1.
2.7. Statistical analysis Initially, normality test was applied to verify if the data had a normal distribution. Leucogram data did not show normal distribution and, thus, they were transformed to logarithmic form. Then, all data of this study were submitted to analysis of variance (oneway ANOVA) followed by Duncan test (P < 0.05). 3. Results 3.1. Disease course We did not observe differences in pre-patent period in group D when compared with group C throughout this study; however it was observed a decreased number of trypomastigotes form in the blood of animals from group D when compared with group C on day 8 PI (P < 0.05). Data are shown in Fig. 1. 3.2. Hematological parameters Full data of hematological parameters are shown in Table 2. Animals infected by T. evansi (Groups C and D) showed a decrease in the erythrocyte count, hematocrit and hemoglobin when compared with uninfected animals. These same differences were not observed in groups A and B (P > 0.05). Animals infected by T. evansi showed an increase in the number of total leukocytes, lymphocytes, and monocytes when compared with uninfected animals (P < 0.05).
Trypanosomes/field (1000x)
a
12 Group C
10 8
Group D
6 4 2 0 1
2
3
4
5
6
7
8
Days post-infection Fig. 1. Average parasitemia of animals infected with Trypanosoma evansi corresponding to Groups C and D (infected). Note: Group D was supplemented with iron (0.6 mL kg−1, subcutaneously).
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Table 2 Mean and standard deviation of haematological variables after iron supplementation on rats experimentally infected by Trypanosoma evansi. Variables
Uninfected
6
−1
Total erythrocytes (×10 μL ) Hematocrit (%) Hemoglobin (g dL−1) MCV (fl) MCHC (%) Total Leukocytes (×103 μL−1) Neutrophils (×103 μL−1) Lymphocytes (×103 μL−1) Monocytes (×103 μL−1) Eosinophils (×103 μL−1)
Infected
Group A (untreated)
Group B (treated)
Group C (untreated)
Group D (treated)
8.1 ± 0.31 43.8 ± 1.6a 13.0 ± 0.47a 54.1 ± 0.4a 30.3 ± 0.5a 5.6 ± 1.3b 1.4 ± 0.8a 4.0 ± 0.9b 0.015 ± 0.034b 0.10 ± 0.12a
8.2 ± 0.27 46.6 ± 1.9a 13.7 ± 0.40a 55.4 ± 2.1ab 30.1 ± 0.4a 6.4 ± 1.9b 2.1 ± 0.9a 4.1 ± 1.1b 0.023 ± 0.053b 0.22 ± 0.31a
6.3 ± 0.41 33.3 ± 3.1c 10.5 ± 1.0c 52.4 ± 1.0b 31.1 ± 0.7a 11.1 ± 3.9a 1.5 ± 0.8a 9.0 ± 3.0a 0.46 ± 0.18a 0.075 ± 0.044a
7.5 ± 0.32b 39.9 ± 3.6b 11.8 ± 0.8b 53.6 ± 1.6ab 31.2 ± 0.8a 10.1 ± 3.1a 1.7 ± 1.3a 7.8 ± 2.4a 0.43 ± 0.28a 0.14 ± 0.07a
a
a
c
Means followed by the same letter in the same line do not differ significantly by the Duncan test (P > 0.05).
uninfected groups (P > 0.05). Total proteins, urea, and creatinine did not differ among groups (P > 0.05).
3.3. Biochemical parameters Full data of biochemical variables, oxidative and antioxidants markers are shown in Table 3. 3.3.1. ADA activity in serum and erythrocytes A decrease on seric ADA activity in animals from group C was observed when compared with uninfected animals (P < 0.05). The iron treatment did not cause significant changes on the activity of ADA in serum of rats from group D when compared with the negative group (P > 0.05). In erythrocytes, a decreased (P < 0.05) activity of E-ADA on group C was observed when compared with the uninfected; however animals from group D showed an increase on ADA activity (P < 0.05), when compared with groups A and group C, both groups that did not receive iron supplementation. 3.3.2. Iron levels No differences on seric iron levels were observed between uninfected groups (Group A and B – P > 0.05), while animals from group C showed decreased iron levels in serum when compared with uninfected rats (P < 0.05); however, animals from group D maintained a similar level of seric iron, as the negative control groups (groups A and B). 3.3.3. ALT No difference was observed on seric levels of ALT between uninfected groups (P > 0.05), but, in group C, an increase on seric levels of ALT (P < 0.05) was observed. In group D, the animals did not show changes in seric levels of ALT when compared with the
3.3.4. AOPP, TOS, FRAP, TAC and NOx Rats from group B showed an increase in AOPP levels compared with uninfected animals (P < 0.05 – Group A). A similar result was observed on groups C and D. Additionally, animals of group D presented decreased TOS levels when compared with other groups (P < 0.05). The negative (A) and positive (C) control groups did not show differences on NOx levels (P < 0.05). However, the opposite situations were observed in animals from groups B and D (supplemented with iron), where decreased levels of NOx were observed, when compared with the other groups (P < 0.05). No difference was observed between uninfected groups on seric FRAP levels (P > 0.05); but animals of group D showed increased FRAP levels when compared with the other groups (P < 0.05). TAC levels did not differ among groups (P > 0.05). 4. Discussion In the present study, rats infected by T. evansi showed reduced values for erythrocytes, hematocrit, and hemoglobin as reported by other authors (França et al., 2011; Menezes et al., 2004; Wolkmer et al., 2009), corroborating with the fact that anemia is a common feature of T. evansi infections (Da Silva et al., 2009; Herrera et al., 2004). Therefore, the reduction of hematological parameters might be a direct consequence of parasitemia, since the parasitemia causes a large number of red blood cells (RBCs) to be removed from circulation by the mononuclear phagocytic system (MPS) in the spleen, bone marrow, and lymph nodes. The removal of a large number of
Table 3 Mean and standard deviation of biochemical variables after iron supplementation on rats experimentally infected by Trypanosoma evansi. Variables
ADA in serum (U L−1) ADA in erythrocytes (U g−1 of Hb) Iron (mg L−1) Total protein (mg dL−1) ALT (U L−1) Urea (mg dL−1) Creatinine (mg dL−1) AOPP (μmol L−1) TOS (μmol H2O2 Equivalent L−1) TAC (×103 mmol Trolox Equivalent L−1) FRAP (×102 μmol L−1) NOx (μmol L−1)
Uninfected
Infected
Group A (untreated)
Group B (treated)
Group C (untreated)
Group D (treated)
6.1 ± 2.0a 0.56 ± 0.07b 3.14 ± 0.21a 8.36 ± 0.09a 52.2 ± 3.1b 43.3 ± 4.7a 0.80 ± 0.09a 38.3 ± 1.0b 23.5 ± 5.9b 1.11 ± 0.05a 5.83 ± 0.77b 104.7 ± 25b
5.8 ± 1.9a 0.64 ± 0.20ab 3.02 ± 0.34a 8.50 ± 0.3a 49.3 ± 3.9b 40.2 ± 5.6a 0.74 ± 0.03a 42.5 ± 0.9a 19.9 ± 5.1b 1.08 ± 0.09a 4.81 ± 1.12b 54.9 ± 27c
3.3 ± 0.8b 0.32 ± 0.1c 1.25 ± 0.18b 8.3 ± 0.5a 64.2 ± 5.1a 44.0 ± 3.4a 0.69 ± 0.04a 43.8 ± 2.1a 25.8 ± 7.2b 1.04 ± 0.12a 5.93 ± 1.5b 128.6 ± 18a
4.8 ± 1.5ab 0.96 ± 0.31a 2.45 ± 0.22ab 8.0 ± 0.4a 49.2 ± 7.3b 39.6 ± 7.8a 075 ± 0.06a 44.8 ± 2.3a 3.4 ± 5.6a 1.04 ± 0.08a 7.69 ± 1.9a 63.4 ± 14c
Note: Animals on groups A and B were not infected, while animals on groups C and D were intraperitoneally infected with trypomastigotes forms of Trypanosoma evansi. Means followed by the same letter in the same line do not differ significantly by the Duncan test (P > 0.05).
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RBCs leads to a drop in packed red cell volume (PCV) to below 25%, reaching to as low as 10% (Matios and Eyob, 2013). It is possible to correlate our hematological findings with the fact that oxidative damage to red blood cells has been related to the generation of free radicals (Vives-Corrons et al., 1995), and that ironreplacement treatment decreases antioxidant stress by regulating enzymatic levels (Isler et al., 2002), which shows that our results are relevant in T. evansi infection. In the current study, iron supplementation led to an increase in AOPP, but it was not able to increase the levels of TOS. Iron is required for the activity of numerous ironand heme-containing proteins, but “free” (i.e. redox active) iron catalyzes the formation of highly toxic reactive oxygen species (ROS) (Graf et al., 1984) that damage lipids, proteins, and DNA (Quinlan et al., 2002), facts that may explain the elevation on AOPP levels. On the other hand, animals infected and treated with iron showed increased FRAP levels, demonstrating additionally the antioxidant strength of iron by reducing Fe3+ to Fe2+ (Benzie and Strain, 1996). The formation of highly toxic ROS due to the iron supplementation, even though is not beneficial to the host, may collaborate to moderate the parasitemia, since the infected animals that were supplemented with iron presented lower parasitemia that the ones infected not supplemented. According to Jain (1986), there are two phases of anemia, which is the most prominent finding in natural trypanosome infections, and the severity of the first phase is proportional to the degree of parasitemia. Thus, iron supplementation may indirectly collaborate to enhance the host response against T. evansi by diminishing the parasitemia. Iron supplementation had also direct effect on NOx levels, since this metal plays an inhibitory role on the expression of inducible NOx synthase (iNOS), which is an inflammatory setting, often accompanied by a large production of ROS gene (Vallabhapurapu and Karin, 2009). This inhibitory action might have collaborated to the reduced levels of NOx observed in the groups supplemented with iron, and also helped to explain the increased levels of ALT in rats infected and not supplemented with iron. Rats infected by T. evansi showed increase level of ALT (Barr, 1991; Monzón and Villavicencio, 1990), but many other studies also demonstrated that hepatocytes express iNOS following exposure to various cytokines such as tumor necrosis factor (TNF), interleukin 1(IL1), interferon gamma (IFNγ), and interleukin six (IL-6) (Adamson and Billings, 1993; Curran et al., 1990; Geller et al., 1993), a pro-inflammatory condition already reported on infections by T. evansi (Paim et al., 2011). Therefore, the presence of free iron into the bloodstream may help to inhibit iNOS, modulating the pro-inflammatory response against the parasite and, also, the concomitant cellular damage to cells and tissues. Similarly to our study, the reduction of ADA activity in serum and erythrocytes of rats infected with T. evansi has been reported previously (Da Silva et al., 2011). Regarding the ADA activity, it is possible to correlate this enzyme directly with the major effect of this parasitic disease, the anemia. It is well known that the main ADA substrate, adenosine, is involved in various functions of T. evansi (Suswam et al., 2003), as well as in the pathogenesis of anemia (Zhang and Xia, 2012; Zhang et al., 2011). In this sense, early in 1990, researchers reported a relationship between iron deficiency and anemia, concluding that iron depletion impairs the ADA activity (Luo et al., 1990). The same authors reported that iron supplementation was able to increase ADA activity in anemic animals, correlating the changes in the enzymatic activity with the iron deficiency. Our results did not show that levels of ADA statistically increased in groups supplemented with iron; however, they showed that the group infected and not supplemented presented reduced activity of ADA (along with reduction in hematologic parameters) in serum and erythrocytes, corroborating with reports of Luo and collaborators (Luo et al., 1990). Based on these results, we can conclude that the iron supplementation was able to provide some effect on the oxidative stress
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imbalance, as well as in the ADA activity modulation. Iron supplementation helped to diminish the parasitemia, and indirectly reduced the establishment of the anemic process; and it was capable of modulating the activity of ADA in serum and erythrocytes, maintaining its levels as equal as in the uninfected groups. Iron supplementation was also able to inhibit iNOS, reducing NOx levels and preventing increase of ALT. Meanwhile, FRAP levels were reduced modulating an oxidative stress situation. However, it was also possible to observe that free iron generated an increase in AOPP levels probably due to protein and DNA damage caused by the association between parasite action and free iron bioavailability.
References Adamson, G.M., Billings, R.E., 1993. Cytokine toxicity and induction of NO sythase activity in cultured mouse hepatocytes. Toxicol. Appl. Pharmacol. 119, 100–107. Anosa, V.O., Kaneko, J.J., 1983. Pathogenesis of Trypanosoma brucei infection in deer mice (Peromyscus maniculatus): hematologic, erythrocyte biochemical, and ironmetabolic aspects. Am. J. Vet. Res. 44, 639–644. Barr, S.C., 1991. American trypanosomiasis in dog. Comp. Cont. Educ. Pract. Vet. 13, 745–752. Benzie, I.F.F., Strain, J.J., 1996. The ferric reducing ability of plasma (FRAP) as a measure of “Antioxidant Power”: The FRAP Assay. Anal. Biochem. 239, 70–76. Calder, P.C., Albers, R., Antoine, J.M., Blum, S., Bourdet-Sicard, R., Ferns, G.A., et al., 2009. Inflammatory disease processes and interactions with nutrition. Br. J. Nutr. 101, 1–45. Colpo, C.B., Monteiro, S.G., Stainki, D.R., 2005. Natural infection by Trypanosoma evansi in dogs. Ciênc. Rural 35, 717–719. Curran, R.D., Billiar, T.R., Stuer, D.J., Ochoa, J.B., Harbrecht, B.G., Flint, S.G., 1990. Multiple cytokines are required to induce hepatocyte nitric oxide production and inhibit total protein synthesis. Ann. Surg. 212, 462–469. Da Silva, A.S., Doyle, R.L., Monteiro, S.G., 2006. Métodos de contenção e confecção de esfregaço sanguíneo para pesquisa de hemoparasitas em ratos e camundongos. FZVA. 13, 83–87. Da Silva, A.S., Costa, M.M., Wolkmer, P., Zanette, R.A., Faccio, L., Gressler, L.T., et al., 2009. Trypanosoma evansi: hematologic changes in experimentally infected cats. Exp. Parasitol. 123, 31–34. Da Silva, A.S., Bellé, L.P., Bitencourt, P.E.R., Souza, V.C.G., Costa, M.M., Oliveira, C.B., et al., 2011. Activity of the enzyme adenosine deaminase in serum, erythrocytes and lymphocytes of rats infected with Trypanosoma evansi. Parasitology 138, 201–208. Doherty, C.P., 2007. Host-pathogen interactions: the role of iron. J. Nutr. 137, 1341–1344. Erel, O., 2004. A novel automated method to measure total antioxidant response against potent free radical reactions. Clin. Biochem. 37, 112–119. Erel, O., 2005. A new automated colorimetric method for measuring total oxidant status. Clin. Biochem. 38, 1103–1111. Feldman, B.F., Zinkl, J.G.A., Jain, N.C., 2000. Schalm’s Veterinary Hematology, fifth edition. Lippincott Williams & Wilkins, Philadelphia, PA, p. 787. França, R.T., Da Silva, A.S., Wolkmer, P., Oliveira, V.A., Pereira, M.E., Leal, M.L.R., et al., 2011. Delta-aminolevulinate dehydratase activity in red blood cells of rats infected with Trypanosoma evansi. Parasitology 138, 1272–1277. Geller, D.A., Nussler, A.K., Di Silvio, M., 1993. Cytokines, endotoxin and glucocorticoids regulate the expression of inducible nitric oxide synthase in hepatocytes. PNAS 90, 522–526. Giusti, G., Gakis, C., 1971. Temperature conversion factors, activation energy, relative substrate specificity and optimum pH of adenosine deaminase from human serum and tissues. Enzyme 12, 417–420. Graf, E., Mahoney, J.R., Bryant, R.G., Eaton, J.W., 1984. Iron-catalyzed hydroxyl radical formation. Stringent requirement for free iron coordination site. J. Biol. Chem. 259, 3620–3624. Hanasand, M., Omdal, R., Norheim, K.B., Goransson, L.G., Brede, C., Jonsson, G., 2012. Improved detection of advanced oxidation protein products in plasma. Clin. Chim. Acta 413, 901–906. Herrera, H.M., Dávila, A.M., Norek, A., Abreu, U.G., Souza, S.S., 2004. Enzootiology of Trypanosoma evansi in Pantanal, Brazil. Vet. Parasitol. 11, 366–369. Hostetter, M.K., Johnson, G.M., 1989. The erythrocyte as investigator of inflammation. Generation of amidated C3 by erythrocyte adenosine deaminase. J. Clin. Invest. 84, 665–671. Iannotti, L.L., Tielsch, J.M., Black, M.M., Black, R.E., 2006. Iron supplementation in early childhood: health benefits and risks. Am. J. Clin. Nutr. 84, 1261–1276. Igbokwe, I.O., 1994. Increased susceptibility of erythrocytes to in vitro peroxidation in acute Trypanosoma brucei infection of mice. Vet. Parasitol. 55, 279–286. Isler, M., Delibas, N., Guclu, M., Gultekin, F., Sutcu, R., Bahceci, M., et al., 2002. Superoxide dismutase and glutathione peroxidase in erythrocytes of patients with iron deficiency anemia: effects of different treatment modalities. Croat. Med. J. 43, 16–19. Jain, N.C., 1986. Schalm’s Veterinary Hematology, fourth edition. Lea & Febiger, Philadelphia, PA. 1221p.
6
N.B. Bottari et al./Experimental Parasitology 147 (2014) 1–6
Luo, C., Chen, J., Liao, Q., Li, Q., Yang, X., Li, Y., 1990. Influence of iron deficiency anemia on development of thymus and spleen and adenosine deaminase activity in rats. Hua Xi Yi Ke Da Xue Xue Bao 21, 63–66. Matios, L., Eyob, E., 2013. Review on camel trypanosomosis (surra) due to Trypanosoma evansi: Epidemiology and host response. J. Vet. Med. Anim. Health 5, 334–343. Menezes, V.T., Queiroz, A.O., Gomes, M.A.M., Marques, M.A.P., Jansen, A.M., 2004. Trypanosoma evansi in inbred and Swiss-Webster mice: distinct aspects of pathogenesis. Parasitol. Res. 94, 193–200. Monzón, C.M., Villavicencio, V.I., 1990. Serum protins inguinea-pigs and horses infected with Trypanosoma evansi (Steel, 1885). Vet. Parasitol. 36, 295–301. Omer, O.H., Mousa, H.M., Al-Wabel, N., 2007. Study on the antioxidant status of rats experimentally infected with Trypanosoma evansi. Vet. Parasitol. 145, 142–145. Paim, F.C., Duarte, M.M., Costa, M.M., Da Silva, A.S., Wolkmer, P., Silva, C.B., et al., 2011. Cytokines in rats experimentally infected with Trypanosoma evansi. Exp. Parasitol. 128, 365–370. Quinlan, G.J., Evans, T.W., Gutteridge, J.M., 2002. Iron and the redox status of the lungs. Free Radic. Biol. Med. 33, 1306–1313. Rodrigues, A., Fighera, R.A., Souza, T.M., Schild, A.L., Soares, M.P., 2005. Surtos de tripanossomíase por Trypanosoma evansi em equinos no Rio Grande do Sul: aspectos epidemiológicos, clínicos, hematológicos e patológicos. Pesqui. Vet. Bras. 25, 239–249. Sampaio, A.F.S., 2012. Effects of Supplementation with Injectable Iron on the Production of ROS by Granulocytes and Oxidative Damage in Cardiac and Pancreatic Tissues of Diabetic Rats (Dissertation, Master in Health and Nutrition). Universidade Federal de Ouro Preto, Ouro Preto, Brazil. 96p. Silva, R.A.M.S., Seidl, A., Ramirez, L., Dávila, A.M.R., 2002. Trypanosoma evansi e Trypanosoma vivax: Biologia, Diagnóstico e controle. Corumbá, Embrapa Pantanal.
Smith, A., 2002. Homeostasis of heme in health and disease: current aspects of the structural biology of heme-protein interactions and of gene regulation. DNA Cell Biol. 21, 245–249. Suswam, E.A., Ross, C.A., Martin, R.J., 2003. Changes in adenosine transport associated with melaminophenyl arsenical (Mel CY) resistance in Trypanosoma evansi: down-regulation and affinity changes of the P2 transporter. Parasitology 127, 543–549. Tatsch, E., Bochi, G.V., Pereira, R.S., Kober, H., Agertt, V.A., De Campos, M.M.A., et al., 2011. A simple and inexpensive automated technique for measurement of serum nitrite/nitrate. Clin. Biochem. 44, 348–350. Tavares, K.C.S., Da Silva, A.K., Wolkmer, P., Monteiro, S.G., Miletti, L.C., 2011. Cryopreservation of Trypanosoma evansi after DEAE-cellulose purificarion: Evaluation of infective parameters. Res. Vet. Sci. 90, 257–259. Vallabhapurapu, S., Karin, M., 2009. Regulation and function of NF-kappaB transcription factors in the immune system. Annu. Rev. Immunol. 27, 693–733. Vives-Corrons, J.L., Miguel-Garcia, A., Pujades, M.A., Miguel-Sosa, A., Cambiazzo, S., Linares, M., et al., 1995. Increased susceptibility of microcytic red blood cells to in vitro oxidative stress. Eur. J. Haematol. 55, 327–331. Weinberg, E.D., 1978. Iron and infection. Microbiol. Rev. 42, 45–66. Wolkmer, P., Silva, A.S., Cargnelutti, J., Costa, M.M., Traesel, C.K., Lopes, S.T.A., et al., 2007. Resposta eritropoética de ratos em diferentes graus de parasitemia por Trypanosoma evansi. Ciênc. Rural 37, 1682–1687. Wolkmer, P., da Silva, A.S., Traesel, C.K., Paim, F.C., Cargnelutti, J.F., Pagnoncelli, M., et al., 2009. Lipid peroxidation associated with anemia in rats experimentally infected with Trypanosoma evansi. Vet. Parasitol. 165, 41–46. Zhang, Y., Xia, Y., 2012. Adenosine signaling in normal and sickle erythrocytes and beyond. Microbes Infect. 14, 863–873. Zhang, Y., Dai, Y., Wen, J., Zhang, W., Grenz, A., Sun, H., et al., 2011. Detrimental effects of adenosine signaling in sickle cell disease. Nat. Med. 17, 79–86.
