Experimental Parasitology 128 (2011) 265–271
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Hymenolepis diminuta: Activity of anti-oxidant enzymes in different parts of rat gastrointestinal tract Danuta I. Kosik-Bogacka a,⇑, Irena Baranowska-Bosiacka b, Iwona Nocen´ b, Katarzyna Jakubowska b, Dariusz Chlubek b a b
Department of Biology and Medical Parasitology, Pomeranian Medical University, Powstancow Wielkopolskich Av. 72, 70-111 Szczecin, Poland Department of Biochemistry and Medical Chemistry, Pomeranian Medical University, Powstancow Wielkopolskich Av. 72, 70-111 Szczecin, Poland
a r t i c l e
i n f o
Article history: Received 14 December 2010 Received in revised form 10 February 2011 Accepted 24 February 2011 Available online 2 March 2011 Keywords: Anti-oxidant enzymes Glutathione Hymenolepidosis Lipid peroxidation Oxidative stress
a b s t r a c t The aim of this study was to assess the intensity of oxidative stress by measuring levels of lipid peroxidation products in the duodenum, jejunum and colon of rats infected with Hymenolepis diminuta and evaluate the effectiveness of protection against oxidative stress by measuring the glutathione levels and activity of anti-oxidant enzymes: superoxide dismutase, catalase, glutathione reductase and glutathione peroxidase. In exposed rats we observed a significant increase of lipid peroxidation products in the duodenum and jejunum. A significant decrease in superoxide dismutase activity in all the examined parts of the digestive tract was observed. Additionally, rats from 16 to 40 days post H. diminuta infection (dpi) had a decreased catalase activity in the colon, while at 60 dpi it increased. The glutathione peroxidase activity increased significantly in the colon at 60 dpi. The increase in glutathione reductase activity was observed in the colon in rats 60 dpi. There was a lack of changes in the levels of glutathione in the duodenum and a significant increase in its concentration in the jejunum and colon from 40 to 60 dpi and from 16 to 40 dpi, respectively. In this study we observed altered activity of anti-oxidant enzymes and glutathione level in experimental hymenolepidosis, as a consequence of oxidative stress. It may indicate a decrease in the efficiency of intestinal protection against oxidative stress induced by the presence of the parasite. The imbalance between oxidant and anti-oxidant processes may play a major role in pathology associated with hymenolepidosis. Ó 2011 Elsevier Inc. All rights reserved.
1. Introduction Hymenolepis diminuta is a non-invasive tapeworm of the small intestine in rodents, mainly rats and mice. Hymenolepis diminuta becomes mature in rats between 16 and 19 days post infection (dpi), and lives until the death of the host. Although infection with H. diminuta is usually asymptomatic, it induces a number of changes in the small intestine of the host. Rats infected with H. diminuta have an increased number of goblet cells (McKay et al., 1990; Starke and Oaks, 2001). Eight days post H. diminuta infection, altered motility within the host small intestine induces increased smooth muscle contractility and a decreased
Abbreviations: ROS, reactive oxygen species; LPO, lipid peroxidation products; SOD, superoxide dismutase; CAT, catalase; GR, glutathione reductase; GPx, glutathione peroxidase; GSH, glutathione; dpi, days post infection; GSSG, oxidized glutathione. ⇑ Corresponding author. Fax: +48 91 4661671. E-mail address:
[email protected] (D.I. Kosik-Bogacka). 0014-4894/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.exppara.2011.02.026
luminal transit rate (Dwinell et al., 1994, 1997); 32 dpi mucosal mastocytosis and smooth muscle hypertrophy have been observed (Dwinell et al., 1998a). The chronic phase of infection by H. diminuta is shown by the disappearance of intestinal villus, inflammatory swelling, lymph system development and erosion in all the sections of the small intestine (Fal and Czaplicka, 1991). These changes concern all segments of the small intestine due to the large size of the parasites and the migration of the tapeworm in the intestine in a circadian migration in response to nutrition of the host (Podesta and Mettrick, 1977). The prevalence of helminths in the digestive tract results in an immediate hypersensitivity reaction in the host (Jarrett and Miller, 1982) and oxidative stress. The generation of reactive oxygen species (ROS): superoxide radicals (O2 ), hydrogen peroxide (H2O2) and hydroxyl radicals (OH) is known to damage various cellular components which leads to the impairment of proteins, DNA and lipid peroxidation products (LPO), which in turn leads to impairment of membrane integrity and adverse changes in cellular metabolism (Halliwell and Gutteridge, 1989; Ohshima and Bartsch, 1994).
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Lipid peroxidation products is a well-established mechanism of cellular injury and is used as an indicator of oxidative stress in various cells and tissues. Polyunsaturated fatty acid peroxides generate malondialdehyde (MDA) upon decomposition, and measurement of MDA concentration has been used as an indicator of LPO (Esterbauer et al., 1991). LPO modifies the properties of cell membranes, thus increasing membrane permeability to H+ ions and other substances, and so reducing the difference in electrical potential on either side of the membrane. LPO also leads to the inhibition of certain membrane enzymes, transport proteins and dysfunction of receptors (mainly muscarinic, adrenergic and histamine receptors) (McConnell et al., 1999). Oxidative stress also modulates electrogenic ion transport in rat jejunum (Orsenigo et al., 2007). Free radicals released by the eosinophils of the host may directly damage the worms. Such reactions have been reported in animals infected with Schistosoma mansoni (Kazura et al., 1985), Trichinella spiralis (Kazura and Aikawa, 1980) and Hymenolepis nana (Niwa and Miyazato, 1996). In addition, many researchers showed that ROS may directly or indirectly participate in worm expulsion (Menzies et al., 2010; Smith, 1989, 1991). ROS also directly affects gastrointestinal mucosa, disrupting the intestinal mucus layer and causing its intensified disintegration (Farhadi et al., 2003). As a result, ROS damages cells, for example by inducing cellular cytokine synthesis and in consequence starting the process of carcinogenesis (Selvam et al., 2009). The effect of ROS on tissues is related to the anti-oxidant protective capacity of a tissue, i.e. in enzymes such as superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR), glutathione peroxidase (GPx) and a non-enzymatic anti-oxidant component, glutathione (GSH). The SOD catalyzes the dismutation of superoxide into hydrogen peroxide which is then converted to H2O by CAT and GPx. Glutathione peroxidase utilizes glutathione, and GSH is then replenished by GR. This glutathione redox reaction is thought to serve as an important mechanism for detoxifying reactive oxygen species. This cycle, however, can be disturbed by a reduction in GSH as well as impairment in the ability to regenerate GSH (Halliwell and Gutteridge, 1989). Moreover, an imbalance of antioxidant enzymes activity might be an important part of the mechanism of pathology of ROS. Especially it concerns the balance between the activities of SOD and CAT (measured by a SOD/CAT ratio), as a too high activity of SOD compared to CAT, in the presence of Fe2+ and Cu2+, may lead to formation of highly reactive hydroxyl radical. When SOD activity is too low, hydroxyl radical may also be produced in a Haber–Weiss reaction (Galle, 2001). The disruption of correct proportion between the activities of these enzymes is adverse especially in increased oxidative stress and the synthesis of a great amount of ROS, including hydrogen peroxide, and may lead to the increased degree and area of tissue impairment (Bartosz, 2003). Glutathione peroxidase has a greater affinity to H2O2, hence its more significant role when concentrations of H2O2 are relatively small (Bartosz, 2003). The decreased activities of GPx or CAT may be mutually compensated. Therefore it is important to know the balance between the activities of SOD and GPx. Changes in the balance between the activities of both enzymes, expressed by the SOD/CAT and SOD/GPx ratios, may suggest a drop in the effectiveness of intestinal protection against excessive levels of hydrogen peroxide. In this study, a continuation of our previous research (KosikBogacka et al., 2010, 2011), we would like to show that oxidative stress is an important part of the mechanism of pathology of hymenolepidosis not only in the habitat of the tapeworm (small intestine), but also in other sites (large intestine). The examinations performed at various phases of invasion may explain if and how the increase in oxidative stress changes during the infection
and if the oxidative stress has some role in the adaptive parasitehost relationship. Hence, the specific aim of our study was to examine the oxidative stress through the measurement of LPO in duodenum, jejunum and colon of rats infected with H. diminuta. We also examined the efficiency of protection against oxidative stress through the measurement anti-oxidant enzyme activities (Zn, Cu-SOD, CAT, GR, and GPx) and GSH concentrations. 2. Materials and methods 2.1. Animals Male Wistar rats (4 months old), were either infected with H. diminuta or uninfected, using a previous method (Kosik-Bogacka et al., 2010). Hymenolepis diminuta WMS il 1 (Stradowski, 1998) was maintained by cyclical passage through flour beetles (Tribolium destructor) and Wistar rats. Infected rats were dosed via stomach tube with 5-cysticercoids in 1 ml of 0.9% NaCl solution. All cysticercoids used for the infections were isolated from T. destructor infected with worm eggs. The animals were housed singly, kept on a 12 h light–dark cycle and were given feed (Murigran, Motycz, Poland) and water ad libitum. The study was approved by the Local Ethics Committee for Scientific Experiments on Animals in Szczecin (Poland). The rats (n = 45) were divided into 5 groups:
control group (n = 7) – uninfected (0 dpi); group I (n = 11) – 8 days post H. diminuta infection (8 dpi); group II (n = 8) – 16 days post H. diminuta infection (16 dpi); group III (n = 12) – 40 days post H. diminuta infection (40 dpi); group IV (n = 7) – 60 days post H. diminuta infection (60 dpi).
Before each experiment, a coproscopic examination of the rats’ faeces was performed to ascertain the presence of the parasites. Uninfected and infected rats were sacrificed by tiopental anaesthesia (Biochemie GmbH, Austria) administered at 100 mg/kg body weight (b.w.) intraperitoneally (i.p.). The rats were weighed, and then their duodenum, jejunum and colon were removed for analysis. The tissues were dissected and their content was removed by gentle rinsing. In the examined rats, 3–5 cestodes were observed (mean 4.5) which is 90% in comparison with the given cysticercoids. We compared biochemical parameters for rats in relation to the number of cestodes (from 3 to 5) and we found no statistically significant differences, and therefore these observations are not included in our work. 2.2. Tissue sample preparation Frozen in liquid nitrogen, tissue samples (duodenum, jejunum or colon) were placed in a thermobox ( 21 °C). A small fragment of the tissue was placed in a metal homogenizator (previously cooled in a container with liquid nitrogen) and poured on 2–3 times with liquid nitrogen; then it was fragmented with a few hammer blows (4–5 times) against a metal mandrel (also previously cooled in a container with liquid nitrogen). Pulverized and frozen samples (volume equal to a 1 mg of protein) were placed with a cooled spoon in an Eppendorf tube containing 500 lL of appropriate buffer (according to commercial enzyme assay kit procedure) and protease inhibitor; previously cooled to a temperature of 4 °C. After a short vortexation, homogenization was carried out with a knife homogenizator for about 15 s. Extract mixtures were centrifuged (3000g for 10 min, at 4 °C) and the supernatant was stored at 80 °C and used for enzymes assay.
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2.3. LPO measurement The product of lipid peroxidation, malondialdehyde (MDA) was measured using a Bioxytech LPO-586 Assay Kit (Oxis Research, Poland), according to manufacturer’s instruction. Prior to homogenization 0.5 M butylated hydroxytoluene (BHT) in acetonitrile was added to prevent sample oxidation during homogenization. PBS was used for sample homogenization. The LPO assay is based on the reaction of chromogenic reagent N-methyl-2-phenylindole with MDA. In brief, 200 lL sample was added to 650 lL of Nmethyl-2-phenylindole in acetonitrile. Next, 150 lL of 12 M HCl was added and samples were incubated at 45 °C for 60 min. After incubation time samples were centrifuged (15,000g, 10 min), and the clear supernatant was transferred to cuvettes. LPO concentration was determined by measuring the absorbance at 586 nm and calculated using standard curves. 2.4. Enzyme assays 2.4.1. SOD activity Total (Cu–Zn and Mn) SOD (EC 1.15.1.1) activity was measured using a Bioxytech SOD-525 Assay Kit (Oxis Research, Poland), according to manufacturer’s procedure. Tissue was washed with 0.9% NaCl containing 0.16 mg/mL heparin prior to homogenization. PBS was used for sample homogenization. The assay is based on SOD mediated increase in the rate of autoxidation of 5,6,6a,11btetrahydro-3,9,10-trihydroxybenzo[c]fluorene in aqueous alkaline solution to yield a chromophore with maximum absorbance at 525 nm. In brief, the 40 lL of sample was incubated for 1 min (37 °C) with 900 lL 2-amino-2-methyl-1,3-propanediol buffer (pH 8.8) containing boric acid and diethylenetriaminepentaacetic acid (DTPA) and with 30 lL 1-methyl-2-vinylpyridinium trifluoromethanesulfonate in HCl. Next 30 lL of 5,6,6a,11b-tetrahydro3,9,10-trihydroxybenzo[c]fluorene was added and reaction mixtures were transferred to cuvettes. SOD activity was determined by measuring the absorbance at 525 nm over time. A 50% inhibition is defined as 1 unit of SOD and the activity was expressed as U/mg protein.
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absorbance of 6.22 106 for NADPH. GPx activity was expressed as U/mg protein. 2.4.4. GR activity The activity of GR (EC 1.6.4.2) was measured using a Bioxytech GR-340 Assay Kit (Oxis Research, Poland), according to manufacturer’s instruction. PBS was used for sample homogenization. The assay is based on the oxidation of NADPH to NADP + catalyzed by limiting concentration of glutathione reductase. One molecule of NADPH is consumed for each molecule of GSSG reduced. In brief, 200 lL sample was added to 400 lL of GSSG. The reaction was initiated by the addition of 400 lL NADPH. Reduction of GSSG is determined indirectly by measurement of the consumption of NADPH, as demonstrated by a decrease in absorbance at 340 nm. One unit of GR activity was defined as the amount of sample required to oxidize 1 lM of NADPH per minute, based on the molecular absorbance of 6.22 106 for NADPH. GR activity was expressed as U/mg protein. 2.5. GSH concentration The concentration of GSH was measured using a Bioxytech GSH-400 Assay Kit (Oxis Research, Poland), according to manufacturer’s instruction. Ice-cold metaphosphoric acid was used for sample homogenization. The samples were kept at 4 °C and used for GSH determination within 1 h. The GSH assay was based on the reaction of 4-chloro-1-methyl-7-trifluromethyl-quinolinium methylsulfate and all mercaptans which were present in the sample. Next, b-elimination reaction under alkaline conditions leads to the formation a chromophoric thione which has a maximal absorbance wavelength at 400 nm. In brief, 40 lL sample was added to 860 lL of potassium phosphate containing diethylenetriamine pentaacetic acid (DTPA). Next, 50 lL of chromogenic reagent and 50 lL 30% NaOH were added, samples were thoroughly mixed and incubated at 25 °C for 10 min in the dark. GSH concentration was determined by measuring the absorbance at 400 nm and calculated using standard curves. 2.6. Protein measurement
2.4.2. CAT activity The activity of CAT (EC 1.11.1.6) was measured using a Bioxytech Catalase-520 Assay Kit (Oxis Research, Poland), according to the manufacturer’s procedure. Phosphate buffer saline (PBS, 20 mM, pH 7.4) was used for sample homogenization. In brief, the 30 lL of sample was incubated 1 min with 500 lL 10 mM H2O2. The reaction was quenched with 500 lL sodium azide. Next, reaction mixtures (20 lL) were transferred to cuvettes and 2 mL of horse radish/chromogen reagent was added. CAT activity was determined by measuring the absorbance at 520 nm using an Alfa 40 (PerkinElmer) spectrophotometer. Catalase activity was expressed as U/mg protein. 2.4.3. GPx activity The activity of cellular GPx (EC 1.11.1.9) was measured using a Bioxytech GPx-340 Assay Kit (Oxis Research, Poland), according to manufacturer’s instruction. Buffer containing 1 mM mercaptoethanol, 5 mM EDTA, 50 mM TRIS–HCl was used for sample homogenization. In brief, 350 lL of sample was added to 350 lL of 50 mM Tris–HCl buffer containing 0.5 mM EDTA, 5 mM NADPH, 42 mM reduced glutathione and 10 U/mL glutathione reductase. The reaction was initiated by the addition of 350 lL 30 mM tertbutyl hydroperoxide, and oxidation of NADPH was detected by monitoring the decrease in absorbance at 340 nm. One unit of GPx activity was defined as the amount of sample required to oxidize 1 lM of NADPH per minute, based on the molecular
Total protein content was determined by Bradford assay (1976) using bovine albumin as a standard. 2.7. Statistical analysis The obtained results were analyzed statistically using Statistica 6.1 software. Arithmetical mean and standard deviation (SD) were calculated for each of the studied parameters. As most of the distributions deviated from the normal distribution (Shapiro–Wilk test), non-parametric tests were used for the analyses. Correlations between the parameters were examined with Spearman’s rank correlation coefficient (Rs). In order to assess the differences between the parameters studied, Kruskal–Wallis Anova followed by Mann–Whitney tests was used. The level of significance was p < 0.05. 3. Results 3.1. The concentration of LPO In uninfected rats, the greatest concentration of LPO was found in duodenum; 38% higher than in the colon and about 44% higher than in the jejunum (Table 1). In rats infected with H. diminuta we found a statistically significant increase in the duodenum LPO concentration in comparison
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Table 1 The lipid peroxidation products concentration (LPO); activity of anti-oxidant enzymes (superoxide dismutase – SOD, catalase – CAT, glutathione reductase – GR, glutathione peroxidase – GPx) and glutathione concentrations (GSH) in the duodenum, jejunum and colon of control and H. diminuta infected rats. Parameters
LPO (mmol/mg protein)
SOD (U/mg protein)
CAT (U/mg protein)
GR (U/mg protein)
GPx (U/mg protein)
GSH (mmol/mg protein)
Tissue
Days post infection (dpi)
Duodenum Jejunum Colon Duodenum Jejunum Colon Duodenum Jejunum Colon Duodenum Jejunum Colon Duodenum Jejunum Colon Duodenum Jejunum Colon
0 (n = 7)
8 (n = 11)
16 (n = 8)
40 (n = 12)
60 (n = 7)
9.03 ± 1.37 6.24 ± 0.88 6.52 ± 1.36 131.80 ± 60.87 140.22 ± 95.84 210.58 ± 68.45 5.93 ± 2.40 5.14 ± 1.49 4.63 ± 1.16 11.90 ± 2.47 0.97 ± 0.42 32.40 ± 9.18 0.65 ± 0.21 0.60 ± 0.18 0.70 ± 0.19 1.03 ± 0.72 0.63 ± 0.19 0.67 ± 0.19
9.72 ± 1.19 17.17 ± 4.51** 5.66 ± 0.54 96.82 ± 44.29 32.21 ± 11.66* 288.54 ± 119.06 5.07 ± 1.1 5.05 ± 1.51 4.68 ± 1.23 10.68 ± 4.97 0.93 ± 0.46 33.81 ± 6.74 0.59 ± 0.25 0.56 ± 0.17 0.76 ± 0.22 1.21 ± 0.71 0.69 ± 0.18 0.68 ± 0.10
10.26 ± 1.58 15.90 ± 2.94** 6.27 ± 1.07 97.78 ± 43.12 81.43 ± 37.87 187.10 ± 95.73 4.56 ± 0.88 4.80 ± 0.65 4.16 ± 0.63** 9.22 ± 4.32 2.51 ± 1.22** 33.53 ± 6.39 0.53 ± 0.15 0.60 ± 0.06 0.61 ± 0.20 0.72 ± 0.14 0.90 ± 0.35 0.99 ± 0.14**
10.70 ± 2.10 19.75 ± 6.65** 6.46 ± 1.49 137.52 ± 57.50 66.23 ± 34.82* 203.71 ± 104.09 4.32 ± 0.96 4.32 ± 0.83 4.19 ± 0.70** 14.46 ± 3.51 3.32 ± 1.37** 37.81 ± 6.09 0.46 ± 0.19 0.44 ± 0.08 0.74 ± 0.51 1.05 ± 0.53 1.14 ± 0.48* 1.11 ± 0.35**
15.16 ± 2.89** 22.36 ± 8.14** 6.49 ± 0.31 53.79 ± 25.10* 11.18 ± 2.84** 118.36 ± 37.81* 6.00 ± 1.25 5.69 ± 1.82 5.52 ± 1.32** 20.84 ± 5.53** 2.93 ± 0.99** 44.64 ± 3.04* 0.54 ± 0.22 0.73 ± 0.16 1.13 ± 0.39* 1.13 ± 0.37 0.81 ± 0.08* 0.81 ± 0.17
Mean values and ± standard deviation are given. n is the size of groups. * Significantly different at p < 0.05 compared to control rats (0 dpi). ** Significantly different at p < 0.005 compared to control rats (0 dpi).
Table 2 The correlation between the lipid peroxidation products concentration (LPO); activity of enzymes (superoxide dismutase – SOD, catalase – CAT, glutathione reductase – GR, glutathione peroxidase – GPx); glutathione concentration (GSH) and time of infection in H. diminuta infected rats. Parameters LPO
SOD
CAT
GR
GPx
GSH
Duodenum Jejunum Colon Duodenum Jejunum Colon duodenum jejunum Colon Duodenum Jejunum Colon Duodenum Jejunum Colon Duodenum Jejunum Colon
Rs
p
+0.65 +0.60 +0.20 0.24 0.51 0.47 0.03 +0.68 +0.04 +0.46 +0.73 +0.47 0.24 +0.04 +0.31 +0.20 +0.41 +0.44
