Cent. Eur. J. Biol. • 7(6) • 2012 • 987-995 DOI: 10.2478/s11535-012-0087-3

Central European Journal of Biology

Enzymatic antioxidant system in the cestode diminuta after chronic infection of the rat

Research Article Hanna Czeczot1, Michał Skrzycki1,*, Monika Majewska1, Małgorzata Podsiad1, Rusłan Salamatin2, Barbara Grytner-Zięcina2

1Chair and Department of Biochemistry, Medical University of Warsaw, 02-097 Warsaw, Poland

2Department of General Biology and Parasitology, Medical University of Warsaw, 02-004 Warsaw, Poland Received 08 May 2012; Accepted 27 July 2012

Abstract: Background: The aim of the present paper was to describe the enzymatic antioxidant system in Hymenolepis diminuta collected from rats exposed to chronic cestode invasion. Methodology: We dissected different tissues of H. diminuta (immature proglottids, genital primordia, hermaphroditic proglottids, early uterus, and gravid uterus) and studied activity of: superoxide dismutases (SOD1 and SOD2), catalase (CAT), glutathione peroxidases (non-Se-dependent GSHPx and Se-dependent GSHPx), glutathione-S- (GST) and glutathione reductase (GSHR), and oxidative stress markers - reduced glutathione (GSH), and the lipid peroxidation level (TBARS). Results: We demonstrated changes in antioxidant activities and levels of oxidative stress markers in different tissues of the parasite. The levels of TBARS and GSH indicate that oxidative stress occurred in tissues located proximal to the intestine wall. Activity of SOD1 was high in all parts of H. diminuta, but the GST activity was the highest of all studied antioxidant . SOD2 activity differed significantly in various parts of H. diminuta. Significant differences were observed for nonSeGSHPx and activity of other GSH-dependent enzymes was generally similar in all the tissues. Conclusions: Our results show that the enzymatic antioxidant system of H. diminuta, allows the parasite to adapt and live under conditions of chronic oxidative stress. It suggests an oxidative-antioxidative balance during interactions between parasite and host.

Keywords: Hymenolepis diminuta • Chronic infection • Enzymatic antioxidant system • Superoxide dismutases • Catalase • Glutathione peroxidases • Glutathione-S-transferase © Versita Sp. z o.o.

1. Introduction antioxidant enzymes in their cells. Nippostrogus dubius expresses two times more and Reactive oxygen species (ROS), which are produced four times more catalase and glutathione reductase by the host organism during parasitic infection, play an than Nippostrogus brasiliensis, and is able to persist for important role as mediators during interaction between several months in rodent. Other helminthes use their the host and the invading parasite [1-3]. Parasites antioxidant enzymes as offensive effectors, by secreting that are characterized by a high degree of antioxidant them externally to damage lymphocytes. Necator enzyme activity may survive in the host body for a very americanus have the ability to release superoxide long period of time. Their antioxidant systems might dismutase, which leads to accumulation of hydrogen be used as a component of the defense against ROS peroxide and damage of host cells [7,8]. generated by the host [4-6]. Certain groups of parasitic worms differ in their Generally helminthes are well equipped in different equipment with antioxidant enzymes. Trematodes types of antioxidant enzymes. Some species control how F. hepatica and S. mansoni both have two superoxide long they persist in the host’s intestine by the amount of dismutase isoenzymes – cytosolic and extracellular.

* E-mail: [email protected] 987 Enzymatic antioxidant system in the cestode Hymenolepis diminuta after chronic infection of the rat

They also have glutathione transferase (GST), but their Warsaw. Infection and isolation were conducted on rats main antioxidant enzyme is peroxiredoxin. Adult forms of following the provisions of the Protection Act and S. mansoni are characterized by low activity of glutathione recommendations of the Local Ethics Committee for peroxidase [9-12]. In cestodes superoxide dismutase, Animal Experiments. glutathione transferase and thioredoxin peroxidase has been described, but there is no evidence for enzymes 2.2. Tissue sample preparation that are able to metabolize hydrogen peroxide [13]. The Hymenolepis diminuta tapeworms (n-3) were cut Nematodes in general have supeoroxide dismutase, into 5 pieces respective to immature proglottids, genital catalase, glutathione peroxidase and transferase, and primordia, hermaphroditic proglottids, early uterus, and peroxiredoxins. There are certain differences in nematodes gravid uterus. families. Intestinal nematodes use GST as a main Tapeworm strobila was divided into tissues, which antioxidant enzyme (Heligmosomoides polygyrus and H. differed in their degree of maturity, on the basis of the contrortus) or peroxiredoxins and GST (A. suum). The analysis conducted on tapeworm strobila of similar length O. volvulus of Filariae family has cytosolic, mitochondrial from the control cultivation that also lasted for a year and extracellular superoxide dismutase, three forms of and a half in the rat. The control tapeworm was fixed GST, and two forms of peroxiredoxins, but does not have and stained with lactocarmin and served as a model to catalase [14-17]. Trichinella species are equipped in establish the length of the particular sections [23,24]. superoxide dismutase as a main antioxidant enzyme and Tissues were homogenized on ice in 10 vol. they also have glutathione [18,19]. of 50 mmol/l Tris-HCl buffer (pH 7.5) containing

Hymenolepis diminuta parasitize various species 1 mmol/l MnCl2, 0.2 mol/l KCl, 0.1% (v/v) Triton X-100 of mammals (including humans), however mainly use and PMSF (phenylmethylsulfonyl fluoride) using a rodents as their definitive hosts. This cestode species Heidolph Diax 900 blender at low speed, five times is very well adapted for living in the adverse and difficult for 2-min periods at 3-min intervals. After 30 min, conditions found in the rat small intestine. Invasions extraction on a magnetic stirrer was carried out and in rats are mainly asymptomatic, they are mostly long the homogenates were centrifuged at 120000×g for term, and the parasite frequently persists in the intestinal 30 min at 4°C. The resulting supernatants were stored lumen up to the natural death of the rat [20-22]. The aim at –80ºC and used for determination of the oxidative of the present study was to determine the enzymatic stress markers: concentration of thiobarbiturate reactive antioxidant system activity in different regions of strobila substances (TBARS) – proportional to the level of lipid of Hymenolepis diminuta after chronic infection in rats. peroxidation, and reduced glutathione (GSH), and the activity of antioxidant enzymes: superoxide dismutase (SOD1 and SOD2), catalase (CAT), selenium- 2. Experimental Procedures dependent (SeGSHPx) and non-selenium-dependent (nonSeGSHPx) glutathione peroxidases, glutathione 2.1. transferase (GST) and glutathione reductase (GSHR). The experiments were conducted on three strobilae of Total protein concentration was also determined in H. diminuta, WSJ strain. This tapeworm strain has been the supernatants. All biochemical parameters were cultured in the laboratory of the Department of General determined in the Chair and Department of Biochemistry, Biology and Parasitology Medical University of Warsaw Medical University of Warsaw. for more than 20 years. Three 6-week old male Lew/ Han strain rats were infected with one cysticercoid of 2.3. Determination of oxidative stress markers H. diminuta per animal. Cysticercoids were obtained The lipid peroxidation level was determined according directly from the intermediate hosts Tribolium destructor to the method described by Ohkawa [25]. This cultured in the same laboratory. During the experiment method employs measurement of thiobarbiturate the tapeworm invasion in all infected rats was confirmed reactive substance concentration (TBARS). Results by coproscopy. Water and food were provided ad libid. were expressed as nmols TBARS/mg of protein. The After 1.5 years rats were put down with Morbitalem, concentration of reduced glutathione (GSH) was dissected and strobilae were collected. Tapeworm determined according to the method described by specimens were then divided into 5 parts: immature Ellman, and Sedlak and Lindsay [26,27]. The GSH proglottids, with genital primordia and hermaphroditic level was expressed as µmols/mg of protein. Protein proglottids, with early and gravid uterus. Biochemical concentration was determined according to the method analyses of all the parts were carried out in the Chair described by Bradford [28], using bovine serum albumin and Department of Biochemistry, Medical University of as a standard.

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2.4. Determination of antioxidant enzyme The level of GSH increased significantly (P≤0.05) in activity successive parts of H. diminuta – the lowest level was SOD1 (EC 1.15.1.1) activity was determined using observed in immature proglottids (5.4 mmol/mg) and the a standard Ransod kit from RANDOX. SOD2 highest in the gravid uterus (15.64 mmol/mg) (Figure 1). (EC 1.15. 1.1) activity was determined according to The lipid peroxidation level was the highest in genital the method described by Beauchamp and Fridovich primordial (4.75 nmol/mg), remarkably high in immature [29] and modified by Oberley and Spitz [30]. Both proglottids (3.2 nmol/mg), distinctly lower in the early methods apply xanthine -xanthine oxidase system for and the gravid uterus, and the lowest in hermaphroditic production of superoxide anion which then reacts with proglottids (0.82 nmol/mg) (Figure 2). The observed 2-(4–odophenyl)-3-(4-nitrophenol)-5-phenyltetrazolium changes were statistically significant. chloride (I.N.T) to form a red formazan dye in the case Activity of SOD1 was relatively high in all parts of SOD1 or reacts with NBT (2,2`-Di-p-nitrofenylo- of H. diminuta, however there were no significant 5,5`-difenylo-3,3`-[3,3`-dimetoxy-4,4`-difenyleno] differences in activity between particular tissues (ranged: – ditetrazolium chloride) giving a blue product. The 0.16-0.18 U/mg). SOD2 activity was significantly higher superoxide dismutase activity is then measured in immature proglottids (0.12 U/mg) and distinctly lower in by degree of inhibition of those reactions. CAT other tissues – the lowest in the early uterus (0.02 U/mg). (EC 1.11.1.6) activity was determined according to the We observed that CAT had just marginal activity method described by Goth [31]. This method relies compared to other studied antioxidant enzymes (0.01 to on measurement of reaction of hydrogen peroxide 0.007 U/mg) (Figure 3). degradation, stopped by addition of ammonium The profile of GSH-dependent enzyme activity is molibdate, which in the presence of H2O2 forms a yellow presented on Figure 4. In successive parts of H. diminuta complex. GSHPxs (EC 1.11.1.9) activity was determined nonSeGSHPx activity decreased significantly – the according to the method described by Wendel [32]. highest level was found in immature proglottids

GSHPxs catalyzes the reduction of H2O2 and various (0.093 U/mg) and the lowest in the gravid uterus organic hydroperoxides using reduced glutathione (0.05 U/mg). SeGSHPx activity was similar in all parts of (GSH) as a donor. The GSHPx assay was carried out H. diminuta (around 0.04 U/mg). by monitoring the oxidation of nicotinamide adenine In immature proglottids GST activity was the highest dinucleotide phosphate (NADPH) in a recycling assay. (0.32 U/mg), whereas in other tissues, the levels were Activity of non-Se-dependent GSHPx and Se-dependent similar (around 0.2 U/mg). The observed differences GSHPx was determined using cumene hydroperoxide were statistically significant. The lowest activity of GSHR

(CHP) and H2O2 as a substrate, respectively. GST was observed in the gravid uterus (0.025 U/mg), while (EC 2.5.1.18) activity was measured according to the in other parts its activity was of similar levels (around method of Habig et al. [33], using chlorodinitrobenzene 0.038 U/mg) (Figure 4). (CDNB) as a substrate. GSHR (EC 1.6.4.2.) activity In all tapeworm tissues, the largest participation in the was assayed using oxidized glutathione (GSSG) as total activity of the antioxidant system was observed for a substrate according to the method described by GST, and SODs, particularly SOD1. The activity of GST Goldberg and Spooner [34]. Specific activity of the seems to be the most stable element of the antioxidant studied enzymes was expressed as U/mg of protein. barrier in all tapeworm tissues. Activity of SOD1 in all tapeworm tissues was higher than SOD2 activity. The 2.5. Statistical analysis largest difference in activity between particular tissues All results are expressed as the mean of three was observed for SOD2. The smallest participation was independent experiments done in triplicate ± S.D. observed for CAT. Significance differences were calculated by the Student-t An important share in total antioxidant enzymatic test. Differences were considered statistically significant system potential was also shown for glutathione if P≤0.05. Statistical evaluation of results was performed peroxidases (nonSeGSHPx and SeGSHPx). One of using the Statistica 9.0 program (StatSoft 9.0). the most important enzymes of the antioxidant barrier influencing its potential is GSHR. Its activity in all studied parts of the tapeworm was generally at the same level. 3. Results It seems that particular parts of the tapeworm are protected against oxidative stress mainly by GST and Concentrations of TBARS, GSH, and activities of SOD1, and then by GSHPx supported by GSHR activity. antioxidant enzymes in H. diminuta isolated from the rat CAT to lesser extent participates in defense of the small intestine are shown in Figures 1, 2, 3 and 4. parasite against the action of ROS.

989 Enzymatic antioxidant system in the cestode Hymenolepis diminuta after chronic infection of the rat

25 Segments: */** 20 */** 15 */**

10 mmol/mg protein

5

0 immature genital primordial hermaphroditic early uterus gravid uterus

* - significant vs immature; ** - significant vs genital primordial (P ≤ 0,05)

Figure 1. GSH level in H. diminuta. In dissected tissues of a tapeworm (immature proglottids, genital primordia, hermaphroditic proglottids, early uterus, and gravid uterus) the level of reduced glutathione (GSH) was determined. It was expressed as µmol/mg of protein.

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4

3 */** */**

2 nmol/mg protein nmol/mg */**

1

0 immature genital primordial hermaphroditic early uterus gravid uterus

*- significant vs immature; ** - significant vs genital primordial (P ≤ 0,05)

Figure 2. Lipid peroxidation level in H. diminuta. The lipid peroxidation level was determined in particular tissues of a tapeworm: immature proglottids, genital primordia, hermaphroditic proglottids, early uterus, and gravid uterus. End products of lipid peroxidation are thiobarbiturate reactive substances, whose concentration (TBARS) was measured and expressed as nmols TBARS/mg of protein.

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0,25 Segments: 0,2

0,15 SOD1 SOD2 CAT 0,1 U/mg proteinU/mg */** 0,05 * */** */**

0 immature genital primordial hermaphroditic early uterus gravid uterus

* - significant vs immature; ** - significant vs genital primordial (P ≤ 0,05)

Figure 3. Superoxide dismutase (SOD) and Catalase (CAT) activity in H. diminuta. Activity of two SOD isoenzymes - cytosolic (SOD1) and mitochondrial (SOD2) – was determined by the degree of inhibition of superoxide anion generation. CAT activity was determined due to rate of hydrogen peroxide degradation. Activity of all enzymes was determined in particular tissues of a tapeworm: immature proglottids, genital primordia, hermaphroditic proglottids, early uterus, and gravid uterus, and expressed as U/mg of protein.

0,4

0,35 Segments:

0,3 * * * * 0,25 nonSeGSHPx SeGSHPx 0,2 GST GSHR U/mg proteinU/mg 0,15

0,1 * * */**/*** */**/*** 0,05

0 immature genital primordial hermaphroditic early uterus gravid uterus

* - significant vs immature; ** - significant vs genital primordial; *** - signifcant vs hermaphroditic (P ≤ 0,05)

Figure 4. Activity of GSH-dependent enzymes in H. diminuta. Selenium-dependent (SeGSHPx) and non-selenium-dependent (nonSeGSHPx) glutathione peroxidases, glutathione tranferase (GST), and glutathione reductase (GSHR) are GSH-dependent enzymes. Activity of these enzymes was determined in particular tissues of a tapeworm: immature proglottids, genital primordia, hermaphroditic proglottids, early uterus, and gravid uterus. Specific activity of the studied enzymes was expressed as U/mg of protein.

4. Discussion mechanisms, the host reacts to the parasites presence and the parasites defensive mechanisms react against Interaction between the parasite and the definitive the host. The mechanisms of antioxidant protection in host is related, among other aspects, to different types parasites are of great importance in studying reactions of adaptations. In the host-parasite system defense of parasite infection and host defense. Successful

991 Enzymatic antioxidant system in the cestode Hymenolepis diminuta after chronic infection of the rat

infection of the host depends on the relationships in tapeworm environment [38]. CAT activity was very between those two systems. Response to infection of low in all tissues of H. diminuta, as this enzyme is the host immunological system relies in large part on rarely found in parasites [6,37,39]. generation of reactive oxygen species (ROS), causing The GSH-dependent enzymes function rather as damage to cellular structures of the parasite [35,36]. detoxifying agents, removing toxic products of oxidative Because parasites live in oxygen-poor environments cell structure damage [40-43]. The most important of they are particularly sensitive to oxidative stress. them in H. diminuta seems to be GST. This enzyme Therefore they have developed different ways to converts toxic metabolites (including products of lipid avoid oxidative damage. Parasites mainly defend peroxidation e.g. 4-hydroxy-2-nonenal) to more soluble, against ROS by means of the antioxidant system. easy to expel compounds, by conjugating them with It consists of nonenzymatic antioxidants such as GSH [44,45]. Therefore GST in H. diminuta might have glutathione, and a set of antioxidant enzymes: SOD1, a protective role against oxidative stress, and toxins SOD2, CAT, SeGSHPx, nonSeGSHPx, GST, GSHR emerging due to processes taking place in the intestinal [6,37]. Therefore exact knowledge about antioxidant lumen (food digestion, bacterial and fungal metabolism, mechanisms of the parasite might contribute to more xenobiotic conversion). Moreover, a low GSH level effective treatment of infected organisms. and increased activity of GST in immature proglottids In this research, experimental hymenolepidosis point to intensive oxidation of tapeworm tissues/cells of rats allowed us to study the antioxidant defense located proximal to the intestine wall. As it was shown mechanisms of the Hymenolepis diminuta tapeworm. in our earlier research, generally, activity of all GSH- Our results showed altered activities of antioxidant dependent enzymes was higher in young tapeworm enzymes and levels of oxidative stress markers in than in old ones, with an exception being GST, whose particular tissues of the parasite (immature proglottids, activity was very low. In old tapeworm the situation is genital primordia, hermaphroditic proglottids, early and opposite – activity of GST was very high [38]. gravid uterus). High activity of GST at the contact site of the The levels of TBARS and GSH indicate oxidative tapeworm strobilae and intestinal epithelium was stress conditions in tissues located proximal to the previously described by Nguyen et al. [46]. Activity of intestine wall (immature proglottids, genital primordia). nonSeGSHPx was the highest in immature proglottids High levels of TBARS and low levels of GSH in those confirming its exposition to oxidative stress. The tissues point to an increased lipid peroxidation level and farther from the intestine wall, the lower the activity of the exhaustion of nonenzymatic antioxidants showing nonSeGSHPx, hence tissues distant from the intestine an exacerbated attack of the host immunological wall are less exposed to oxidative stress. We obtained system. However, the level of GSH was high in early analogous results in our previous research which and gravid uterus, which probably plays a role in compared the antioxidant system of young and old protection of tapeworm eggs against oxidative stress. H. diminuta during rat infection [47]. Comparing this result with studies on early infection, in In all tissues of the tapeworm, generally low young tapeworm a higher lipid peroxidation and lower activity of SeGSHPx and very low CAT activity point to

GSH level was observed (except of tissues close to scavenging of H2O2 via other antioxidant mechanisms. intestine wall). This suggests the adaptation of old It is well known that peroxiredoxins (PRx) exist in tapeworm to oxidative stress during infection lifespan numerous parasitic species. Peroxiredoxins are [38]. well described. There are 2 types of these enzymes The high level of SOD1 activity and the alterations differing in protein structure: peroxiredoxins – with 1 of SOD2 activity observed in all H. diminuta tissues, cys at the , and thioredoxins with 2 cys in indicates adaptation of parasite metabolism to their active site. The third type is thioredoxin peroxidase oxidative stress conditions and response to the attack using thioredoxin as a donor of reducing thiols. All of the host immunological system. The highest activity these types of enzymes convert organic peroxides or

of SOD2 was observed in immature proglottids, which hydrogen peroxide into less toxic compounds, which have the greatest exposure to oxidative stress due could alternatively be very reactive [6,37,48,49]. to an inflammatory state of the host. Study on young GSHR activity was similar in all tapeworm tissues, tapeworm has shown that activity of SOD1 and SOD2 except for a low level in the gravid uterus. This might is the highest in immature proglottids, while in other be a result of a high GSH level in this tissue, which tissues it is similar. This suggests that SOD1 activity provides effective antioxidant defense. It seems that forms stable antioxidant defense, but SOD2 is more there is no need of increased reductive activity of able to respond to actual conditions of oxidative stress GSHR.

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5. Conclusions of GSH, and a high lipid peroxidation level in these tissues. Other parts must only be protected against The enzymatic antioxidant system of Hymenolepis toxins descending from the intestine lumen, and it diminuta is one of the most important mechanisms seems that this pivotal role is performed by GST (the which allow the parasite to adapt to a hostile highest of all studied antioxidant enzymes). Finally, in environment. Our results show that in experimental H. diminuta, the potential of the antioxidant system hymenolepidosis, activity of the enzymatic antioxidant mainly depends on the activity of GST, SOD1 and system of the studied H. diminuta allows the parasite SOD2. Catalase is of minor importance due to its very to adapt and live under conditions of chronic oxidative low activity. stress. Tissues located proximal to the intestine wall are Observed changes of oxidative stress marker levels the most exposed to oxidative stress related with the and the particular activity of antioxidant enzymes in the inflammatory state of the host. Therefore we observed H. diminuta tapeworm during the chronic infection of the highest activity of a number of antioxidant enzymes rats indicates the development of an adaptive parasite (GST, SOD2, nonSeGSHPx,), the lowest concentration – host relationship.

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