Blood Levels of Oxidant/Antioxidant Parameters in Rats Infected with Toxoplasma gondii

Somayeh Bahrami; Ali Shahriari; Mehdi Tavalla; Somayeh Azadmanesh; Hossein Hamidinejat

doi:10.1155/2016/8045969

. 2016 Sep 26;2016:8045969. doi: 10.1155/2016/8045969

Blood Levels of Oxidant/Antioxidant Parameters in Rats Infected with Toxoplasma gondii

Somayeh Bahrami ^1,^*, Ali Shahriari ², Mehdi Tavalla ³, Somayeh Azadmanesh ¹, Hossein Hamidinejat ¹

PMCID: PMC5056297 PMID: 27746857

Abstract

Toxoplasmosis is a common parasitic infection in the world. Since increased free radicals and oxidative stress are reported in many parasitic diseases the purpose of the present study was to evaluate the oxidative stress in acute and chronic toxoplasmosis. RH strains of Toxoplasma tachyzoites were used in the present study. Twenty-five female rats were infected with the parasite while 25 other rats were as the control group that received normal saline. Zero-, 5-, 7-, 10-, and 45-day postinfection (DPI) blood samples were taken. Some parameters related to oxidant and antioxidants such as antioxidant enzymes, malondialdehyde, and total antioxidant capacity were measured. On day 7 after infection, GPX activity and GSH level were significantly increased and in the mentioned day the amount of total antioxidant capacity was significantly reduced. In other cases, there were no significant differences between the groups in different days. Overall, based on the results it seems that, on day 7 after infection, in infected rats responses to oxidative stress were triggered and led to decrease of total antioxidant capacity. Furthermore, glutathione was increased to cope with stress. It seems that probably antioxidant defense system entered the infection to the chronic phase and changed the parasites stage.

1. Introduction

Toxoplasma gondii is an obligate intracellular protozoan parasite responsible for toxoplasmosis, a disease that affects many mammals including man [1, 2]. Infection of an immunocompetent individual is usually asymptomatic; however, infection of immunocompromised individuals or congenital infection of a fetus can lead to debilitating or life-threatening illness. Infection generally occurs through either ingestion of oocysts shed in the faeces of a cat, which is the definitive host, or ingestion of viable tissue-cysts in undercooked meat. When primary infection occurs during pregnancy, congenital transmission could occur. Initial infection and acute disease are characterized by the presence of fast-replicating tachyzoites. Around 10–14 days after infection, tachyzoites differentiate into bradyzoites that replicate more slowly and form cysts in tissues throughout the body. Tissue cysts are long-lived and not associated with disease. However, in people with immunodeficiency, such as AIDS or malignancies, rupture of tissue cysts and the transformation of bradyzoites to tachyzoites result in disease reactivation. Congenitally infected individuals are also at risk of repeated disease reactivation, most notably in their brain and eyes, although the reason(s) for this has not been established. Recent investigations indicate that parasitic infections with high tolerance of the host are the result of defense mechanisms which include enhanced generation of reactive oxygen species (ROS) [3, 4]. Cells and biological fluids from an antioxidant defense system aid at suppression of ROS generation and prevention of ROS reactions with cellular components. A balance between oxidants and antioxidant is known to exist under physiological conditions. However, even small changes in oxidant or/and antioxidant levels may disturb its balance and leads to oxidative stress. This situation becomes dangerous when the antioxidant system is unable to prevent oxidative reactions triggered by ROS and directed oxidative modification of lipids, proteins, and DNA [5–7]. Free radicals are produced continuously by normal metabolic processes, but their rate of production increases during certain parasitic infections.

Stage conversion between tachyzoite and bradyzoite forms of Toxoplasma is associated with morphological and molecular biological changes, including stage-specific antigen expression and alterations to metabolism [8]. Different studies confirmed that alkaline medium, heat shock protein, and acid conditions can induce stage conversion in vitro. These methods appear to rely on stressing the parasites. Having these facts in mind, the present study was designed to compare the antioxidant profile of rats during acute and chronic toxoplasmosis and investigate probable role of oxidative stress in Toxoplasma gondii tachyzoite-bradyzoite interconversion.

2. Materials and Methods

2.1. Parasite

The virulent RH strain of T. gondii was obtained from Jundishapur University of Medical Sciences, Ahvaz, Iran. Tachyzoites of this strain were collected by serial intraperitoneal passages in BALB/c mice. Parasites (1 × 10⁵) were inoculated in the mice, and after 72 hours, tachyzoites were provided by repeated flushing of the peritoneal cavity by phosphate buffered saline (PBS). Tachyzoites were then harvested and centrifuged at 200 ×g for 5 min at room temperature to remove peritoneal cells and cellular debris. The supernatants were collected and centrifuged at 800 ×g for 10 min. The pellets, enriched with parasite tachyzoites, were recovered with PBS and used in the experiments [9].

2.2. In Vivo Study

Fifty female Wistar rats, 8 to 10 weeks old, approximately 250–280 g, were purchased from laboratory animal breeding council (Jundishapur University of Medical Science, Ahvaz, Iran). They were housed in a room under controlled temperature (24 ± 2°C), lighting (12 h light/dark cycle), and relative humidity 40–70% conditions. During experiment, all rats had free access to water and standard rat chow. After 1 week acclimation, firstly modified agglutination test was used to ensure rats were not already infected with Toxoplasma, and then they were divided randomly into two equal groups: The infected and control groups were each inoculated intraperitoneally with 1 × 10⁷ organisms [10] and normal saline, respectively. On days 0, 5, 7, 10, and 45 after infection, five rats from each group were euthanized in a glass desiccator jar for open-drop anesthesia with chloroform following standard animal ethics guidelines of Iran. Blood samples were obtained by cardiac puncture into sterile vacuum tubes with and without anticoagulant (EDTA). Serum was separated by centrifuge and stored at −20°C until use. During the present study, the animals were handled according to the recommendation of the Ethics Committee, Shahid Chamran University of Ahvaz, Ahvaz, Iran (Ethical Approval number 29/6/94, 30 Sept. 2014). Acute and chronic toxoplasmosis of was confirmed by PCR and modified agglutination test (MAT), respectively.

2.3. DNA Extraction and Polymerase Chain Reaction (PCR) Amplification

Induction of acute toxoplasmosis was confirmed with PCR in infected rats on days 5, 7, and 10 after infection. DNA was extracted from whole blood using a genomic DNA purification kit (CinnaGen, Iran). For detection of T. gondii DNA, primers targeting the G529 gene were selected from the literature [11]. PCR reactions included a negative control, consisting of the reaction mix and 2 μL of DNase/RNase-free water and a positive control that consisted of a DNA sample from the tachyzoites of T. gondii. All PCR were performed in a 25 μL reaction containing 12.5 μL Taq DNA polymerase master mix Red (Amplicon, Denmark), 1 μM primers, and 50 ng DNA templates. PCR cycling included an initial denaturation at 94°C for 4 min, followed by 30 cycles of denaturation at 94°C for 50 s, annealing at 57°C for 50 s, and extension at 72°C for 60 s. This was followed by a final extension at 72°C for 5 min. PCR products were electrophoresed in 1.5% agarose (SinaClon Bioscience, Iran) in Tris-acetic acid-EDTA (TAE) buffer, stained with Green Safe stain (SinaClon Bioscience, Iran), and visualized under ultraviolet light. Positive samples showed a band of approximately 400 bp.

2.4. Modified Agglutination Test

To ensure that chronic toxoplasmosis occurred in rats on day 45 after infection, MAT was used as described by Desmonts and Remington and Dubey and Desmonts [12, 13]. The sera were diluted twofold (1 : 20 to 1 : 320) with phosphate buffered saline containing 0.2 M 2-mercaptoethanol and 50 μL of each dilution was put in a well of 96 U-bottom ELISA plates. Thereafter, 50 μL of the whole formalin-preserved T. gondii tachyzoites was added to each serum dilution. The wells were then mixed thoroughly by pipetting up and down several times, covered, and then incubated at 37°C overnight. The test was considered positive when a layer of agglutinated parasites was formed in wells at dilutions of 1 : 20 or higher. Positive and negative controls were included in each test.

2.5. Oxidant/Antioxidant Assessment

The determination of the SOD activity was based on the generation of superoxide radicals produced by xanthine and xanthine oxidase, which react with 2-(4-iodophenyl)-3-(4-nitrophenol)-5-phenyltetrazolium chloride to form a red formazon dye. Briefly, 300 μL of mixed substrate was added to 200 μL of diluted hemolysates. The samples were mixed well and 75 μL xanthine oxidase was added to reactions. The absorbance was measured at 505 nm and the SOD activity was then calculated according to the manufacturer's instruction (Ransod®-Randox Lab, Antrim, UK) and expressed as U/mL.

Glutathione peroxidase activity was determined based on the fact that GPX catalyzed the oxidation of glutathione by cumene hydroperoxide. In presence of the glutathione reductase and nicotinamide adenine dinucleotide phosphate (NADPH), the oxidized glutathione was immediately converted to the reduced form with concomitant oxidation of NADPH to NADP⁺. To evaluate GPX activity in hemolysates 10 μL of samples was mixed with 500 μL reagent R1 and 20 μL cumene R2. The absorbance was measured at 340 nm and the GPX activity was then calculated according to the manufacturer's instruction (Ransel®-Randox Lab, Antrim, UK). The enzymes activities were expressed as U/mL.

The procedure to estimate the reduced glutathione (GSH) level followed the method described by Ellman [14]. In this method thiols react with Ellman's reagent (5,5′-dithiobis-(2-nitrobenzoic acid) or DTNB), cleaving the disulfide bond to give 2-nitro-5-thiobenzoate (TNB⁻), which ionizes to the TNB²⁻ dianion in water at neutral and alkaline pH.

To evaluate GSH level in samples, 15 μL of hemolysates was mixed with 260 μL assay buffer (0.1 M sodium phosphate and 1 mM EDTA, pH: 8) and 5 μL Ellman reagents. Samples were incubated for 15 min at room temperature and the TNB²⁻ formation was quantified in a spectrophotometer by measuring the absorbance of visible light at 412 nm. Absorbance values were compared with a standard curve generated from standard curve from known GSH.

Catalase activity was determined spectrophotometrically by the method of Koroliuk et al. [15]. Briefly, 10 μL of sample was incubated with 100 μmol/mL of H₂O₂ in 0.05 mmol/L Tris-HCl buffer pH = 7 for 10 min. The reaction was terminated by rapidly adding 50 μL of 4% ammonium molybdate. Yellow complex of ammonium molybdate and H₂O₂ was measured at 410 nm. One unit of catalase activity was defined as the amount of enzyme required to decompose 1 μmol H₂O₂ per min.

Total antioxidant capacity of serum was measured according to the method of Benzie and Strain [16]. Briefly, a working solution of FRAP (ferric reducing antioxidant power) was provided by mixing buffer acetate with TPTZ solution in HCl. After that FeCl₃ was added and mixed. 8 μL of serum and 240 μL of mentioned working solution were mixed and incubated for 10 min at room temperature. The optical density of samples was measured at 532 nm. Total antioxidant capacity was expressed as mmol/L.

Malondialdehyde levels in samples were measured using the thiobarbituric acid reaction method of Placer et al. [17]. Quantification of the thiobarbituric acid reactive substances was determined at 532 nm by comparing the absorption to the standard curve of MDA equivalents generated by acid-catalyzed hydrolysis of 1,1,3,3-tetramethoxypropane. To measure MDA level, a working solution containing 15% trichloroacetic acid, 0.375% thiobarbituric acid, and 0.25 N hydrochloric acid was prepared. For each sample, 250 μL serum and 500 μL working solution were mixed and placed in boiling water for 10 min. After cooling the samples were centrifuged at 3000 rpm for 10 min. Finally 200 μL of each supernatant was transferred to microplates and the optical density of samples was measured at 535 nm. The values of MDA were expressed as μmol/L.

2.6. Statistical Analysis

The mean value and standard error were calculated for each group of measurements. The Kolmogorov-Smirnov test was used to test the normal distribution of the data before statistical analysis was performed. Statistical analyses were conducted with the general linear model procedure of SAS for Windows version 9.1 (SAS, 1998) to determine if variables differed between groups. Whenever significant differences were found, mean values were compared by the Tukey test. A probability value of less than 0.05 was considered significant.

3. Results

After induction of experimental toxoplasmosis, acute toxoplasmosis was confirmed by PCR. In all the samples obtained 5 and 7 days after infection, DNA of Toxoplasma was detected in blood. Ten days after infection except for the two samples others were positive. Although in two samples DNA was not detected they were serologically positive. The accuracy of experimental chronic toxoplasmosis induction (samples obtained 45 days after infection) was serologically checked out. In all the samples complete carpet of agglutinated organisms was seen and considered as positive.

The antioxidant enzymes activities, total antioxidant capacity, and MDA level in blood of experimentally infected rats were compared to those of noninfected.

Changes of SOD activity in different days in infected (p = 0.91) and uninfected group (p = 0.62) were not significant. Also after comparing SOD activity in infected and uninfected groups on the days 0 (p = 0.81), 5 (p = 0.36), 7 (p = 0.75), 10 (p = 0.35), and 45 (p = 0.80) after infection, significant changes were not found (Table 1).