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Hymenolepis diminuta: Experimental studies on the antioxidant system with short and long term infection periods in the rats
Experimental Parasitology 129 (2011) 158–163
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Hymenolepis diminuta: Experimental studies on the antioxidant system with short and long term infection periods in the rats Michał Skrzycki a, Monika Majewska a, Małgorzata Podsiad a, Hanna Czeczot a, Rusłan Salamatin b, Joanna Twarowska b, Barbara Grytner-Zie˛cina b,⇑ a b
Chair and Department of Biochemistry, Medical University of Warsaw, Banacha 1, 02-097 Warsaw, Poland Department of General Biology and Parasitology, Medical University of Warsaw, Chałubinskiego 5, 02-004 Warsaw, Poland
a r t i c l e
i n f o
Article history: Received 25 September 2010 Received in revised form 15 June 2011 Accepted 28 June 2011 Available online 20 July 2011 Keywords: Hymenolepis diminuta Infection Oxidative stress Antioxidant enzymes GSH TBARS
a b s t r a c t Many helminths cause long-lasting infections, living for several years in mammalian hosts reflecting a well balanced coexistence between host and parasite. There are many possible explanations as to how they can survive for lengthy periods. One possibility is their antioxidant systems, which can serve as defence mechanisms against host-generated oxygen radicals. Therefore, the aim of this experimental study was to examine the antioxidant system in Hymenolepis diminuta during short (1.5 months young tapeworms) and long (1.5 years old tapeworms) term infection in the rat small intestine. The strobilae of H. diminuta tapeworms (14 young and three old) were divided into three pieces: the anterior part, containing the genital primordiae in the immature segments; the medial part, containing the early uterus in the mature, hermaphroditic proglottids and the terminal part with the mature gravid uterus in the gravid segments. Supernatants of these fragments were used for determination of markers of oxidative stress: concentration of thiobarbiturate reactive substances (TBARS) and of reduced glutathione (GSH), and the activity of antioxidant enzymes: superoxide dismutase (SOD1 and SOD2), catalase (CAT), glutathione peroxidases (GSHPxs), glutathione transferase (GST) and glutathione reductase (GSHR). The results indicated changes in levels of oxidative stress markers and antioxidant enzyme activity in both the young and old forms of H. diminuta. Relatively high activity of SOD (particularly in the anterior part of young tapeworms) was observed, as was increased activity of total GSHPx and a relatively high concentration of GSH in all parts of the tapeworms. These are caused by exposure to increased amount of ROS, which are produced during the inflammatory state. Due to the high activity of antioxidant enzymes, the anterior section of young and old tapeworms is equipped with a very effective antioxidant system. Old organisms also effectively resist oxidative stress due to reduced levels of lipid peroxidation and the high activity of GST, all of which suggest good adaptation to the hostile environment in the host’s intestine. Ó 2011 Elsevier Inc. All rights reserved.
1. Introduction It has been known for a long time that many helminths develop a well balanced coexistence with their mammalian hosts in which they can live for many years, despite the rather hostile conditions that develop in response to their presence in the host organs and tissues. There are many different explanations as to how they can survive for long periods of time. Among them, recent studies have focused on the antioxidant systems, which have evolved in these parasites and may serve as defence mechanisms against the hostgenerated oxygen radicals accompanying parasitic inflammation (Bennet et al., 1993; Henkle-Dührsen and Kampkötter, 2001; Chiumiento and Bruschi, 2009). Hymenolepis diminuta is a parasite of the small intestine capable of parasitizing a wide variety of rodents (mostly mice and rats); it ⇑ Corresponding author. E-mail address: [email protected] (B. Grytner-Zie˛cina). 0014-4894/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.exppara.2011.06.014
has also been recorded from other mammals including man (Ashford and Crewe, 2003; Barrett, 2009). Experimental infections with H. diminuta in rats can last for the duration of the host’s life (our unpublished data). This suggests good adaptation of this tapeworm to its host, and it is possible that the underlying mechanism is based on a very effective antioxidant system. Therefore, the aim of this experimental study was to examine the antioxidant system in H. diminuta during short and long term infections in the rat small intestine. 2. Material and methods 2.1. Parasites This experimental study was carried out using 12 Lew/Han male rats, having been approved by the Local Ethic Committee for Animal Experiments. The experimental animals, were subdivided into four groups, including two control, non infected groups, with three rats
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in each group. The two experimental groups, infected with H. diminuta, comprised: (1) a heavy infection group of three rats, each infected with five cysticercoids and examined 1.5 months after infection; and (2) one light infection group of three rats, each infected with a single cysticercoid, in order to increase their chance of survival for a long period of time, examined 1.5 years after infection. Fourteen young tapeworms were collected from the intestines of the three heavily infected rats 1.5 months after infection. From the intestines of the three rats examined 1.5 year after infection three tapeworms were collected. From each tapeworm the following fragments of their strobilae were collected: (I) The anterior part, containing the genital primordial in the immature segments; (II) The medial part, containing the early uterus in the mature, hermaphroditic proglottids; (III) The terminal part with the mature gravid uterus in the gravid segments. The parasitological work was conducted in the Department of General Biology and Parasitology, while the biochemical work was carried out in the Department of Biochemistry of the Medical University of Warsaw. 2.2. Biochemical analysis 2.2.1. Samples preparation The strobilae of H. diminuta tapeworms (14 young and three old) were divided into three pieces corresponding to the anterior, medial and terminal parts. All the sections were homogenized on ice in 10 V of 50 mmol/l Tris–HCl buffer (pH 7.5) containing 1 mmol/l MnCl2, 0.2 mol/l KCl, 0.1% (v/v) Triton X-100 and PMSF (phenylmethylsulfonyl fluoride) using a Heidolph Diax 900 blender at low speed, five times for 2-min periods at 3-min intervals. After 30 min extraction on a magnetic stirrer, the homogenates were centrifuged at 120,000g for 30 min at 4 °C. The supernatants were used for determination of the markers of oxidative stress: concentration of thiobarbiturate reactive substances (TBARS) – proportional to the level of lipid peroxidation, the concentration of reduced glutathione (GSH), and the activity of antioxidant enzymes: superoxide dismutase (SOD1 and SOD2), catalase (CAT), glutathione peroxidases (GSHPxs), glutathione transferase (GST) and glutathione reductase (GSHR). Total protein concentration was also determined in supernatants. 2.2.2. Determination of oxidative stress markers The lipid peroxidation level was determined according to the method described by Ohkawa et al. (1979). This method employs measurement of the concentration of thiobarbiturate reactive substances (TBARS). The pink chromogen produced by the reaction of thiobarbituric acid with malondialdehyde (MDA) and other secondary products of lipid peroxidation were estimated at 532 nm. Results were expressed as nmols TBARS/mg of protein. The concentration of reduced glutathione (GSH) was determined according to the method described by Ellman (1959); Sedlak and Lindsay (1968). This method is based on the formation of the colored product, which is formed by the reaction of GSH with DTNB (5,50 -dithio-bis-[2-nitrobenzoic acid]). The formation of GSH-DTNB conjugate was monitored by the change of absorbance at 412 nm. The concentration of GSH was expressed as lmols/mg of protein. Protein concentration was determined according to the method described by Bradford (1976), using bovine albumin as a standard.
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2.2.3. Determination of antioxidant enzymes activity Superoxide dismutases CuZnSOD (SOD1) and MnSOD (SOD2). SOD1 (EC 1.15.1.1) activity was determined using standard Ransod kit from RANDOX. This method employs xanthine oxidase and xanthine to generate superoxide radicals, which subsequently react with 2-(4-idophenyl)-3-(4-nitrophenol)-5-phenyltetrazolium chloride (INT) and forms a red formazan dye which was measured at 505 nm. Superoxide dismutase neutralizing O2 inhibits this reaction. SOD2 (EC 1.15. 1.1) activity was determined according to the method described by Beauchamp and Fridovich (1971) and modified by Oberley and Spitz (1984). It is based on inhibition of superoxide radical generation, during the reaction of xanthine oxidase with xanthine. Superoxide radical reduces nitroblue tetrazolium chloride (NBT) to a violet complex, which was measured at 560 nm. Catalase (CAT) (EC 1.11.1.6) activity was determined according to the method described by Góth (1991). The enzymatic reaction was stopped with ammonium molybdate and the yellow complex of molybdate and hydrogen peroxide was measured at 405 nm. Glutathione peroxidases (GSHPxs) (EC 1.11.1.9) activity was determined according to the method described by Paglia and Valentine (1967); Wendel (1981). GSHPxs assay was carried out by monitoring the oxidation of nicotinamide adenine dinucleotide phosphate (NADPH) in a recycling assay. Total activity of GSHPx (tot. GSHPx) was determined using cumene hydroperoxide (CHP) as a substrate. Selenium-dependent glutathione peroxidase (SeGSHPx) activity was measured using H2O2 as a substrate. In the presence of glutathione reductase (GSHR) and NADPH, the oxidized glutathione is immediately converted to the reduced form with a concomitant oxidation of NADPH to NADP+. Reduction in the absorbance of NADPH at 340 nm was measured. GlutathioneS-transferase (GST; EC 2.5.1.18) activity was measured according to the method of Habig et al. (1974) using chlorodinitrobenzene (CDNB) as a substrate. Formation of GSH–CDNB conjugate was monitored by the change of absorbance at 340 nm. Glutathione reductase (GSHR; EC 1.6.4.2.) activity was assayed by using oxidized glutathione (GSSG) as a substrate according to the method described by Golberg and Spooner (1983). GSHR activity assay was based on changes of the absorbance at 340 nm due to oxidation/reduction of NADPH/NADP+. Activity of studied enzymes was expressed as U/mg of protein. 3. Statistical analysis All the results are expressed as means of three independent experiments each carried out in triplicate ± S.D. Significance of differences was calculated by Student-t and Mann-Whitney U tests. Differences were considered statistically significant if p 6 0.05. Statistical evaluation of the results was conducted using the Statistica 6.0 programme (StatSoft 6.0). 4. Results Concentrations of TBARS and reduced glutathione (GSH), activities of antioxidant enzymes in young and old H. diminuta isolated from rat colon are shown in Figs. 1–4. 4.1. Antioxidant system of young H. diminuta tapeworms The lipid peroxidation level, expressed as TBARS, was lowest in the anterior part of H. diminuta (1.58 ± 0.97 nmol/mg protein). The highest level of lipid peroxidation was observed in the terminal part (12.80 ± 4.2 nmol/mg protein). In the medial segment, the TBARS level was increased 8.1-fold in comparison to the level in the anterior and decreased threefold compared to the level in the
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A
SOD1
b, c
SOD2
nmol/mg protein
CAT Anterior segment
tot-GSHPx
Superior segment
Se-GSHPx
Posterior segment
GST GSH-R
b
a
a
a
B SOD1
Young tapeworm
Old tapeworm
SOD2
Fig. 1. Lipid peroxidation level in young and old H. diminuta. Abbreviations: a – significant versus young H. diminuta (p 6 0.05); b – significant versus anterior part of young H. diminuta (p 6 0.05); c – significant versus medial part of young H. diminuta (p 6 0.05); d – significant versus anterior part of old H. diminuta (p 6 0.05); e – significant versus medial part of old H. diminuta (p 6 0.05).
CAT tot-GSHPx
b
Se-GSHPx
b
b
GST
b Anterior segment
Superior segment
b
Posterior segment
a, d, e
b
C
c nmol/mg protein
GSH-R
SOD1 SOD2
a, d
b, c
b
CAT
c
tot-GSHPx Se-GSHPx
b, c
a
GST
c b, c
c
GSH-R
Fig. 3. Activity of antioxidant enzymes in particular parts of young H. diminuta (A – anterior part; B – medial part; C – terminal part). Abbreviations see Fig. 1. Young tapeworm
Old tapeworm
Fig. 2. Level of GSH in young and old H. diminuta. Abbreviations see Fig. 1.
terminal part. All these differences were statistically significant (p 6 0.05). In young H. diminuta GSH levels were similar in parts I and III, however in part II a significant reduction in GSH level was observed compared to the levels in parts I and III (Figs. 1 and 2). In all parts (particularly in the anterior) high level of SOD1, SOD2 and tot. GSHPx and low levels of CAT, SeGSHPx, GST and GSHR activities were observed. CAT activity was the lowest of all the measured enzymes. In the medial part the enzymes had significantly lower activity as compared to the activity observed in the anterior and terminal parts (p 6 0.05). The activity of SOD1 was higher in all parts of young tapeworms (Fig. 3).
4.2. Antioxidant system of old H. diminuta tapeworms TBARS and GSH levels (0.80 ± 0.24 nmol/mg protein and 6.20 ± 1.90 lmol/mg protein, respectively) were lower in the anterior part in comparison with the levels measured in all other parts. The levels of TBARS and GSH (1.60 ± 0.35 nmol/mg protein and 15.64 ± 4.89 lmol/mg protein, respectively) were highest in the terminal part (p 6 0.05). All observed differences were statistically significant (p 6 0.05). (Figs. 1 and 2). In all parts of old tapeworms high activity of GST and SOD1 was observed, whereas activities of SOD2, CAT and GSH-dependent
enzymes (tot. GSHPx, SeGSHPx, GST and GSHR) were significantly decreased, with CAT activity being the lowest. The activity of GST was highest among all enzymes that were assessed. The activities of tot. GSHPx, SeGSHPx, and GSHR were almost unchanged in all three parts. The activities of all antioxidant enzymes assessed was highest in the anterior part (p 6 0.05). (Fig. 4). 4.3. Comparison of antioxidant defenses in young and old H. diminuta tapeworms living in alimentary tract of rats The levels of TBARS and GSH were significantly lower in old than in young H. diminuta. tapeworms (p 6 0,05). Activities of most of antioxidant enzymes (SODs, GSHPxs, and GSHR) were about 10fold lower in old compared with young tapeworms (p 6 0.05). In old parasites the activity of GST was about twofold higher (p 6 0.05) but the activity of CAT in young and old parasites was generally very low (Figs. 1–4). 5. Discussion Effective antioxidant systems constitute important mechanisms for supporting the survival of parasites in their hosts, serving as a defense against free radicals released during the inflamed state induced by the host’s immune system (Andreassen et al., 1999; Mosser, 2003). While infecting a host, parasites may induce a host immune response, which may include activation of eosinophils, neutrophils and macrophages, cytokines release and production of
M. Skrzycki et al. / Experimental Parasitology 129 (2011) 158–163
A
a SOD1 SOD2
a
CAT tot-GSHPx Se-GSHPx
a
a
a
B
GST
a
a, d
GSH-R
SOD1 SOD2 CAT
a
tot-GSHPx Se-GSHPx
a
a
a
a
a
GST GSH-R
C
a, d
SOD1 SOD2 CAT
a
tot-GSHPx Se-GSHPx
a
a a,d,e
a
a
GST GSH-R
Fig. 4. Activity of antioxidants enzymes in particular parts of old H. diminuta. Abbreviations see Fig. 1.
antibodies (IgE) (Hoffmann et al., 2001; Maizels and Yazdanbakhsh, 2003; Dzik, 2006). These particular leukocytes are capable of producing large amounts of reactive oxygen species (ROS) able to directly destroy parasite cells. The precursor of all ROS is the superoxide anion (O2), which is generated in leukocytes by NADPH oxidase during single electron oxidation of molecular oxygen (Klion and Nutman, 2004; Shin et al., 2009) and undergoes the spontaneous reaction of dismutation yielding hydrogen peroxide (H2O2). With ferrous or copper ions hydrogen peroxide might undergo the Fenton reaction which produces the most reactive ROS, the hydroxyl radical (HO⁄), which then reacts with proteins by changing their biological properties and activities, with DNA by causing mutations and with membrane lipids by initiating the lipid peroxidation process. Damage to cellular structures leads to impairment of metabolism and eventually to cell death (Halliwall, 1996; Bergendi et al., 1999; Droge, 2002; Das and White, 2002). Therefore, to survive in such a hostile environment in the host, parasites developed strategies to avoid death. One of these is the antioxidant system comprising a number of antioxidant enzymes (Callahan et al., 1988; Chiumiento and Bruschi, 2009). The key antioxidant enzyme is superoxide dismutase (SOD) which scavenges superoxide anions and produces hydrogen peroxide. There are three isoenzymes of SOD: cytosolic (CuZnSOD), mitochondrial (MnSOD), and extracellular (ECSOD) (Cross and Jones, 1991; James, 1994; Fridovich, 1995). Another important enzyme is catalase (CAT) which degrades hydrogen peroxide into water and oxygen. However, its activity is induced only in high concentrations of H2O2 (Callahan et al., 1990; Sies, 1993; Bruschi and Lucchi, 2001). The second enzyme responsible for degradation of H2O2 is
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selenium-dependent glutathione peroxidase (SeGSHPx) which belongs to the family of enzymes referred to as GSH-dependent enzymes (Hayes and McLellan, 1999; Brigelius-Flohe, 1999). These enzymes use reduced glutathione (GSH) as a co-substrate, a donor of hydrogen, producing oxidized glutathione (GSSG) (Sies, 1999; Flohe et al., 1999). Another enzyme from this family – total glutathione peroxidase (tot. GSHPx) reduces toxic organic peroxides (LOOH) produced during lipid peroxidation, thus blocking this reaction. The end products of the lipid peroxidation process (malondialdehyde MDA, hydroxynonenal HNE) are very toxic to the cells (Pastore et al., 2002). To detoxify them, there is another GSH-dependent enzyme – glutathione-S-transferse (GST), which uses GSH to reduce and detoxify endogenous and exogenous toxic substances and metabolize the products derived from oxidative stress in the cells (Brophy and Barre, 1990; Hayes et al., 2005). Another important enzyme belonging to the GSH-dependent family is glutathine reductase (GSHR), which regenerates reduced glutathione from its oxidized form (Hayes and McLellan, 1999; Sies, 1999). These enzymes represent the first line of defense and have a fundamental role in limiting damage of biological macromolecules and cellular membranes (Sies, 1993). This complicated network of antioxidant enzymes works effectively only if the balance between their activities is maintained. Too high or too low activity of each or any of the antioxidant enzymes can cause an increase in the ROS with resultant generation of even more toxic derivatives. During infection the immune system of the host acts to impair the balance between generation and removal of ROS in parasite cells, whereas parasite metabolism attempts to maintain this balance in order to facilitate parasite survival (Henkle-Dührsen and Kampkötter, 2001; Chiumiento and Bruschi, 2009). In our study we observed changes in oxidative stress markers levels, and in antioxidant enzyme activity in the anterior, medial, and terminal parts of H. diminuta. The anterior part of the tapeworm is most exposed to the action of host defense systems due to its direct contact with the intestinal wall, through the attachment of the scolex to the mucosa. Therefore it has to be equipped with very effective antioxidant systems. In young tapeworms we observed low level of oxidative stress indicated by reduced lipid peroxidation and relatively high concentrations of GSH. Very high activity of SOD isoenzymes and tot. GSHPx indicate effective removal of O2 and possible lipid peroxides (LOOH). Low levels of CAT and SeGSHPx activity indicate degradation of H2O2 by other enzymatic pathways. In parasites this is performed mainly by peroxiredoxins (PRx), which are capable of metabolizing hydrogen peroxide into water and molecular oxygen (McGonigle et al., 1998; Seo et al., 2000). Low activity of GST suggests a limited need for detoxification of toxins other than ROS. In the anterior part of old tapeworms we observed similar profiles of antioxidant defense. Reduced lipid peroxidation may still be an indicator of low oxidative stress; however we observed low levels of GSH indicating depletion of its reserves, and this may be connected with the unchanged activity levels of GSHR. In contrast, high activity of SOD isoenzymes effectively scavenges superoxide anion, the activity of GSHPxs being unchanged, therefore hydrogen peroxide may not be sufficiently removed, and contribute to damage of cellular structures. However, the significantly increased activity of GST in old tapeworms points to effective removal of the toxic products of proteins and lipid oxidation, and various electrophilic substances. In the medial part of young tapeworms, increased TBARS levels and decreased GSH levels indicate intensification of oxidative stress. Decreased activities of SOD isoenzymes (twofold), CAT, GSHPxs and GSHR possibly contribute to this process. It seems that due to the relatively long distance from the intestinal wall there is
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no need to maintain a high activity of antioxidant enzymes. Only the activity of GST is increased. This probably provides sufficient protection of the cells of the medial part by detoxification of lipid peroxidation products and exogenous toxins. GST is efficient in this protective effect, since it can catalyze the conjugation of GSH with various electrophilic substances. During the reactions mediated by GST, the conjugates that are produced are actively secreted by the cells of tapeworms, with subsequent depletion of GSH. In old tapeworms we also observed reduced levels of oxidative stress indicated by the very low activity of MnSOD and unchanged activity of other enzymes and oxidative stress markers. The terminal part of young tapeworms was characterized by the highest level of both TBARS and GSH. This indicates intensification of oxidative stress, which is probably balanced by high concentrations of GSH. Antioxidant enzyme activities in young tapeworms were higher than in the medial but lower than in the anterior parts. In the terminal part of old tapeworms, we also found very high levels of lipid peroxidation, with simultaneous high levels of GSH, which may be related to the observed low activity of antioxidant enzymes (CuZnSOD, CAT, tot. GSHPx, GST) indicating diminution of antioxidant system in old tapeworms. In all parts of both, young and old tapeworms, we found very low activity of CAT. The observed values of CAT activity in our study are close to the limit of sensitivity of the analytical method. Our results are in line with observations of numerous authors that CAT activity is not found usually in most parasitic species (Callahan et al., 1988; HenkleDührsen and Kampkötter, 2001). Comparison of the activities of antioxidant enzymes in young and old tapeworms showed about 10-fold lower levels in all parts of old worms. Only the GST activity was higher, perhaps indicating diminution of antioxidant system in old tapeworms as a result of depletion and oxidative damage of relevant enzymes. However, we observed decreased level of TBARS in old tapeworms, which may suggest that the antioxidant system of old tapeworms has lower activity due to the weaker response of the immune system of the host. During infection, parasites are able to suppress defensive mechanisms of the host and adapt to oxidative stress conditions. After a long persistence of tapeworm in the host, this suppression cause the silencing of specific components of the host response to parasitic infection.
6. Conclusions The results reported in this paper indicate differences in the antioxidant systems of both the young and old forms of H. diminuta. Changes in the levels of oxidative stress markers and antioxidant enzymes activity indicate that tapeworms are exposed to high levels of ROS, a result of oxidative stress caused by the inflammatory state that is associated with parasitic infections. Due to the high activity of antioxidant enzymes, the anterior segments of young and old tapeworms are equipped with very effective antioxidant systems. To survive in the conditions of the inflamed intestinal state H. diminuta has adapted its metabolism to cope with the oxidative stress. This is mainly reflected in relatively high activity of the key antioxidant enzyme – SOD, particularly in the anterior segment of young tapeworms. Also the increased activity of total GSHPx points to an effective scavenging of organic peroxides, which are toxic derivatives of ROS. Relatively high concentrations of GSH in all segments of tapeworms also indicate good antioxidant defenses. On the other hand, decreased level of lipid peroxidation and high activity GST in old tapeworms indicate that old organisms effectively act against oxidative stress. This suggests finely tuned adaptation to the hostile environment.
Acknowledgments We are grateful to Professor Jerzy M. Behnke, School of Biology, University of Nottingham, UK for his kind help in the linguistic correction of the final version of this paper.
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Experimental Parasitology journal homepage: www.elsevier.com/locate/yexpr
Hymenolepis diminuta: Experimental studies on the antioxidant system with short and long term infection periods in the rats Michał Skrzycki a, Monika Majewska a, Małgorzata Podsiad a, Hanna Czeczot a, Rusłan Salamatin b, Joanna Twarowska b, Barbara Grytner-Zie˛cina b,⇑ a b
Chair and Department of Biochemistry, Medical University of Warsaw, Banacha 1, 02-097 Warsaw, Poland Department of General Biology and Parasitology, Medical University of Warsaw, Chałubinskiego 5, 02-004 Warsaw, Poland
a r t i c l e
i n f o
Article history: Received 25 September 2010 Received in revised form 15 June 2011 Accepted 28 June 2011 Available online 20 July 2011 Keywords: Hymenolepis diminuta Infection Oxidative stress Antioxidant enzymes GSH TBARS
a b s t r a c t Many helminths cause long-lasting infections, living for several years in mammalian hosts reflecting a well balanced coexistence between host and parasite. There are many possible explanations as to how they can survive for lengthy periods. One possibility is their antioxidant systems, which can serve as defence mechanisms against host-generated oxygen radicals. Therefore, the aim of this experimental study was to examine the antioxidant system in Hymenolepis diminuta during short (1.5 months young tapeworms) and long (1.5 years old tapeworms) term infection in the rat small intestine. The strobilae of H. diminuta tapeworms (14 young and three old) were divided into three pieces: the anterior part, containing the genital primordiae in the immature segments; the medial part, containing the early uterus in the mature, hermaphroditic proglottids and the terminal part with the mature gravid uterus in the gravid segments. Supernatants of these fragments were used for determination of markers of oxidative stress: concentration of thiobarbiturate reactive substances (TBARS) and of reduced glutathione (GSH), and the activity of antioxidant enzymes: superoxide dismutase (SOD1 and SOD2), catalase (CAT), glutathione peroxidases (GSHPxs), glutathione transferase (GST) and glutathione reductase (GSHR). The results indicated changes in levels of oxidative stress markers and antioxidant enzyme activity in both the young and old forms of H. diminuta. Relatively high activity of SOD (particularly in the anterior part of young tapeworms) was observed, as was increased activity of total GSHPx and a relatively high concentration of GSH in all parts of the tapeworms. These are caused by exposure to increased amount of ROS, which are produced during the inflammatory state. Due to the high activity of antioxidant enzymes, the anterior section of young and old tapeworms is equipped with a very effective antioxidant system. Old organisms also effectively resist oxidative stress due to reduced levels of lipid peroxidation and the high activity of GST, all of which suggest good adaptation to the hostile environment in the host’s intestine. Ó 2011 Elsevier Inc. All rights reserved.
1. Introduction It has been known for a long time that many helminths develop a well balanced coexistence with their mammalian hosts in which they can live for many years, despite the rather hostile conditions that develop in response to their presence in the host organs and tissues. There are many different explanations as to how they can survive for long periods of time. Among them, recent studies have focused on the antioxidant systems, which have evolved in these parasites and may serve as defence mechanisms against the hostgenerated oxygen radicals accompanying parasitic inflammation (Bennet et al., 1993; Henkle-Dührsen and Kampkötter, 2001; Chiumiento and Bruschi, 2009). Hymenolepis diminuta is a parasite of the small intestine capable of parasitizing a wide variety of rodents (mostly mice and rats); it ⇑ Corresponding author. E-mail address: [email protected] (B. Grytner-Zie˛cina). 0014-4894/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.exppara.2011.06.014
has also been recorded from other mammals including man (Ashford and Crewe, 2003; Barrett, 2009). Experimental infections with H. diminuta in rats can last for the duration of the host’s life (our unpublished data). This suggests good adaptation of this tapeworm to its host, and it is possible that the underlying mechanism is based on a very effective antioxidant system. Therefore, the aim of this experimental study was to examine the antioxidant system in H. diminuta during short and long term infections in the rat small intestine. 2. Material and methods 2.1. Parasites This experimental study was carried out using 12 Lew/Han male rats, having been approved by the Local Ethic Committee for Animal Experiments. The experimental animals, were subdivided into four groups, including two control, non infected groups, with three rats
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in each group. The two experimental groups, infected with H. diminuta, comprised: (1) a heavy infection group of three rats, each infected with five cysticercoids and examined 1.5 months after infection; and (2) one light infection group of three rats, each infected with a single cysticercoid, in order to increase their chance of survival for a long period of time, examined 1.5 years after infection. Fourteen young tapeworms were collected from the intestines of the three heavily infected rats 1.5 months after infection. From the intestines of the three rats examined 1.5 year after infection three tapeworms were collected. From each tapeworm the following fragments of their strobilae were collected: (I) The anterior part, containing the genital primordial in the immature segments; (II) The medial part, containing the early uterus in the mature, hermaphroditic proglottids; (III) The terminal part with the mature gravid uterus in the gravid segments. The parasitological work was conducted in the Department of General Biology and Parasitology, while the biochemical work was carried out in the Department of Biochemistry of the Medical University of Warsaw. 2.2. Biochemical analysis 2.2.1. Samples preparation The strobilae of H. diminuta tapeworms (14 young and three old) were divided into three pieces corresponding to the anterior, medial and terminal parts. All the sections were homogenized on ice in 10 V of 50 mmol/l Tris–HCl buffer (pH 7.5) containing 1 mmol/l MnCl2, 0.2 mol/l KCl, 0.1% (v/v) Triton X-100 and PMSF (phenylmethylsulfonyl fluoride) using a Heidolph Diax 900 blender at low speed, five times for 2-min periods at 3-min intervals. After 30 min extraction on a magnetic stirrer, the homogenates were centrifuged at 120,000g for 30 min at 4 °C. The supernatants were used for determination of the markers of oxidative stress: concentration of thiobarbiturate reactive substances (TBARS) – proportional to the level of lipid peroxidation, the concentration of reduced glutathione (GSH), and the activity of antioxidant enzymes: superoxide dismutase (SOD1 and SOD2), catalase (CAT), glutathione peroxidases (GSHPxs), glutathione transferase (GST) and glutathione reductase (GSHR). Total protein concentration was also determined in supernatants. 2.2.2. Determination of oxidative stress markers The lipid peroxidation level was determined according to the method described by Ohkawa et al. (1979). This method employs measurement of the concentration of thiobarbiturate reactive substances (TBARS). The pink chromogen produced by the reaction of thiobarbituric acid with malondialdehyde (MDA) and other secondary products of lipid peroxidation were estimated at 532 nm. Results were expressed as nmols TBARS/mg of protein. The concentration of reduced glutathione (GSH) was determined according to the method described by Ellman (1959); Sedlak and Lindsay (1968). This method is based on the formation of the colored product, which is formed by the reaction of GSH with DTNB (5,50 -dithio-bis-[2-nitrobenzoic acid]). The formation of GSH-DTNB conjugate was monitored by the change of absorbance at 412 nm. The concentration of GSH was expressed as lmols/mg of protein. Protein concentration was determined according to the method described by Bradford (1976), using bovine albumin as a standard.
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2.2.3. Determination of antioxidant enzymes activity Superoxide dismutases CuZnSOD (SOD1) and MnSOD (SOD2). SOD1 (EC 1.15.1.1) activity was determined using standard Ransod kit from RANDOX. This method employs xanthine oxidase and xanthine to generate superoxide radicals, which subsequently react with 2-(4-idophenyl)-3-(4-nitrophenol)-5-phenyltetrazolium chloride (INT) and forms a red formazan dye which was measured at 505 nm. Superoxide dismutase neutralizing O2 inhibits this reaction. SOD2 (EC 1.15. 1.1) activity was determined according to the method described by Beauchamp and Fridovich (1971) and modified by Oberley and Spitz (1984). It is based on inhibition of superoxide radical generation, during the reaction of xanthine oxidase with xanthine. Superoxide radical reduces nitroblue tetrazolium chloride (NBT) to a violet complex, which was measured at 560 nm. Catalase (CAT) (EC 1.11.1.6) activity was determined according to the method described by Góth (1991). The enzymatic reaction was stopped with ammonium molybdate and the yellow complex of molybdate and hydrogen peroxide was measured at 405 nm. Glutathione peroxidases (GSHPxs) (EC 1.11.1.9) activity was determined according to the method described by Paglia and Valentine (1967); Wendel (1981). GSHPxs assay was carried out by monitoring the oxidation of nicotinamide adenine dinucleotide phosphate (NADPH) in a recycling assay. Total activity of GSHPx (tot. GSHPx) was determined using cumene hydroperoxide (CHP) as a substrate. Selenium-dependent glutathione peroxidase (SeGSHPx) activity was measured using H2O2 as a substrate. In the presence of glutathione reductase (GSHR) and NADPH, the oxidized glutathione is immediately converted to the reduced form with a concomitant oxidation of NADPH to NADP+. Reduction in the absorbance of NADPH at 340 nm was measured. GlutathioneS-transferase (GST; EC 2.5.1.18) activity was measured according to the method of Habig et al. (1974) using chlorodinitrobenzene (CDNB) as a substrate. Formation of GSH–CDNB conjugate was monitored by the change of absorbance at 340 nm. Glutathione reductase (GSHR; EC 1.6.4.2.) activity was assayed by using oxidized glutathione (GSSG) as a substrate according to the method described by Golberg and Spooner (1983). GSHR activity assay was based on changes of the absorbance at 340 nm due to oxidation/reduction of NADPH/NADP+. Activity of studied enzymes was expressed as U/mg of protein. 3. Statistical analysis All the results are expressed as means of three independent experiments each carried out in triplicate ± S.D. Significance of differences was calculated by Student-t and Mann-Whitney U tests. Differences were considered statistically significant if p 6 0.05. Statistical evaluation of the results was conducted using the Statistica 6.0 programme (StatSoft 6.0). 4. Results Concentrations of TBARS and reduced glutathione (GSH), activities of antioxidant enzymes in young and old H. diminuta isolated from rat colon are shown in Figs. 1–4. 4.1. Antioxidant system of young H. diminuta tapeworms The lipid peroxidation level, expressed as TBARS, was lowest in the anterior part of H. diminuta (1.58 ± 0.97 nmol/mg protein). The highest level of lipid peroxidation was observed in the terminal part (12.80 ± 4.2 nmol/mg protein). In the medial segment, the TBARS level was increased 8.1-fold in comparison to the level in the anterior and decreased threefold compared to the level in the
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A
SOD1
b, c
SOD2
nmol/mg protein
CAT Anterior segment
tot-GSHPx
Superior segment
Se-GSHPx
Posterior segment
GST GSH-R
b
a
a
a
B SOD1
Young tapeworm
Old tapeworm
SOD2
Fig. 1. Lipid peroxidation level in young and old H. diminuta. Abbreviations: a – significant versus young H. diminuta (p 6 0.05); b – significant versus anterior part of young H. diminuta (p 6 0.05); c – significant versus medial part of young H. diminuta (p 6 0.05); d – significant versus anterior part of old H. diminuta (p 6 0.05); e – significant versus medial part of old H. diminuta (p 6 0.05).
CAT tot-GSHPx
b
Se-GSHPx
b
b
GST
b Anterior segment
Superior segment
b
Posterior segment
a, d, e
b
C
c nmol/mg protein
GSH-R
SOD1 SOD2
a, d
b, c
b
CAT
c
tot-GSHPx Se-GSHPx
b, c
a
GST
c b, c
c
GSH-R
Fig. 3. Activity of antioxidant enzymes in particular parts of young H. diminuta (A – anterior part; B – medial part; C – terminal part). Abbreviations see Fig. 1. Young tapeworm
Old tapeworm
Fig. 2. Level of GSH in young and old H. diminuta. Abbreviations see Fig. 1.
terminal part. All these differences were statistically significant (p 6 0.05). In young H. diminuta GSH levels were similar in parts I and III, however in part II a significant reduction in GSH level was observed compared to the levels in parts I and III (Figs. 1 and 2). In all parts (particularly in the anterior) high level of SOD1, SOD2 and tot. GSHPx and low levels of CAT, SeGSHPx, GST and GSHR activities were observed. CAT activity was the lowest of all the measured enzymes. In the medial part the enzymes had significantly lower activity as compared to the activity observed in the anterior and terminal parts (p 6 0.05). The activity of SOD1 was higher in all parts of young tapeworms (Fig. 3).
4.2. Antioxidant system of old H. diminuta tapeworms TBARS and GSH levels (0.80 ± 0.24 nmol/mg protein and 6.20 ± 1.90 lmol/mg protein, respectively) were lower in the anterior part in comparison with the levels measured in all other parts. The levels of TBARS and GSH (1.60 ± 0.35 nmol/mg protein and 15.64 ± 4.89 lmol/mg protein, respectively) were highest in the terminal part (p 6 0.05). All observed differences were statistically significant (p 6 0.05). (Figs. 1 and 2). In all parts of old tapeworms high activity of GST and SOD1 was observed, whereas activities of SOD2, CAT and GSH-dependent
enzymes (tot. GSHPx, SeGSHPx, GST and GSHR) were significantly decreased, with CAT activity being the lowest. The activity of GST was highest among all enzymes that were assessed. The activities of tot. GSHPx, SeGSHPx, and GSHR were almost unchanged in all three parts. The activities of all antioxidant enzymes assessed was highest in the anterior part (p 6 0.05). (Fig. 4). 4.3. Comparison of antioxidant defenses in young and old H. diminuta tapeworms living in alimentary tract of rats The levels of TBARS and GSH were significantly lower in old than in young H. diminuta. tapeworms (p 6 0,05). Activities of most of antioxidant enzymes (SODs, GSHPxs, and GSHR) were about 10fold lower in old compared with young tapeworms (p 6 0.05). In old parasites the activity of GST was about twofold higher (p 6 0.05) but the activity of CAT in young and old parasites was generally very low (Figs. 1–4). 5. Discussion Effective antioxidant systems constitute important mechanisms for supporting the survival of parasites in their hosts, serving as a defense against free radicals released during the inflamed state induced by the host’s immune system (Andreassen et al., 1999; Mosser, 2003). While infecting a host, parasites may induce a host immune response, which may include activation of eosinophils, neutrophils and macrophages, cytokines release and production of
M. Skrzycki et al. / Experimental Parasitology 129 (2011) 158–163
A
a SOD1 SOD2
a
CAT tot-GSHPx Se-GSHPx
a
a
a
B
GST
a
a, d
GSH-R
SOD1 SOD2 CAT
a
tot-GSHPx Se-GSHPx
a
a
a
a
a
GST GSH-R
C
a, d
SOD1 SOD2 CAT
a
tot-GSHPx Se-GSHPx
a
a a,d,e
a
a
GST GSH-R
Fig. 4. Activity of antioxidants enzymes in particular parts of old H. diminuta. Abbreviations see Fig. 1.
antibodies (IgE) (Hoffmann et al., 2001; Maizels and Yazdanbakhsh, 2003; Dzik, 2006). These particular leukocytes are capable of producing large amounts of reactive oxygen species (ROS) able to directly destroy parasite cells. The precursor of all ROS is the superoxide anion (O2), which is generated in leukocytes by NADPH oxidase during single electron oxidation of molecular oxygen (Klion and Nutman, 2004; Shin et al., 2009) and undergoes the spontaneous reaction of dismutation yielding hydrogen peroxide (H2O2). With ferrous or copper ions hydrogen peroxide might undergo the Fenton reaction which produces the most reactive ROS, the hydroxyl radical (HO⁄), which then reacts with proteins by changing their biological properties and activities, with DNA by causing mutations and with membrane lipids by initiating the lipid peroxidation process. Damage to cellular structures leads to impairment of metabolism and eventually to cell death (Halliwall, 1996; Bergendi et al., 1999; Droge, 2002; Das and White, 2002). Therefore, to survive in such a hostile environment in the host, parasites developed strategies to avoid death. One of these is the antioxidant system comprising a number of antioxidant enzymes (Callahan et al., 1988; Chiumiento and Bruschi, 2009). The key antioxidant enzyme is superoxide dismutase (SOD) which scavenges superoxide anions and produces hydrogen peroxide. There are three isoenzymes of SOD: cytosolic (CuZnSOD), mitochondrial (MnSOD), and extracellular (ECSOD) (Cross and Jones, 1991; James, 1994; Fridovich, 1995). Another important enzyme is catalase (CAT) which degrades hydrogen peroxide into water and oxygen. However, its activity is induced only in high concentrations of H2O2 (Callahan et al., 1990; Sies, 1993; Bruschi and Lucchi, 2001). The second enzyme responsible for degradation of H2O2 is
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selenium-dependent glutathione peroxidase (SeGSHPx) which belongs to the family of enzymes referred to as GSH-dependent enzymes (Hayes and McLellan, 1999; Brigelius-Flohe, 1999). These enzymes use reduced glutathione (GSH) as a co-substrate, a donor of hydrogen, producing oxidized glutathione (GSSG) (Sies, 1999; Flohe et al., 1999). Another enzyme from this family – total glutathione peroxidase (tot. GSHPx) reduces toxic organic peroxides (LOOH) produced during lipid peroxidation, thus blocking this reaction. The end products of the lipid peroxidation process (malondialdehyde MDA, hydroxynonenal HNE) are very toxic to the cells (Pastore et al., 2002). To detoxify them, there is another GSH-dependent enzyme – glutathione-S-transferse (GST), which uses GSH to reduce and detoxify endogenous and exogenous toxic substances and metabolize the products derived from oxidative stress in the cells (Brophy and Barre, 1990; Hayes et al., 2005). Another important enzyme belonging to the GSH-dependent family is glutathine reductase (GSHR), which regenerates reduced glutathione from its oxidized form (Hayes and McLellan, 1999; Sies, 1999). These enzymes represent the first line of defense and have a fundamental role in limiting damage of biological macromolecules and cellular membranes (Sies, 1993). This complicated network of antioxidant enzymes works effectively only if the balance between their activities is maintained. Too high or too low activity of each or any of the antioxidant enzymes can cause an increase in the ROS with resultant generation of even more toxic derivatives. During infection the immune system of the host acts to impair the balance between generation and removal of ROS in parasite cells, whereas parasite metabolism attempts to maintain this balance in order to facilitate parasite survival (Henkle-Dührsen and Kampkötter, 2001; Chiumiento and Bruschi, 2009). In our study we observed changes in oxidative stress markers levels, and in antioxidant enzyme activity in the anterior, medial, and terminal parts of H. diminuta. The anterior part of the tapeworm is most exposed to the action of host defense systems due to its direct contact with the intestinal wall, through the attachment of the scolex to the mucosa. Therefore it has to be equipped with very effective antioxidant systems. In young tapeworms we observed low level of oxidative stress indicated by reduced lipid peroxidation and relatively high concentrations of GSH. Very high activity of SOD isoenzymes and tot. GSHPx indicate effective removal of O2 and possible lipid peroxides (LOOH). Low levels of CAT and SeGSHPx activity indicate degradation of H2O2 by other enzymatic pathways. In parasites this is performed mainly by peroxiredoxins (PRx), which are capable of metabolizing hydrogen peroxide into water and molecular oxygen (McGonigle et al., 1998; Seo et al., 2000). Low activity of GST suggests a limited need for detoxification of toxins other than ROS. In the anterior part of old tapeworms we observed similar profiles of antioxidant defense. Reduced lipid peroxidation may still be an indicator of low oxidative stress; however we observed low levels of GSH indicating depletion of its reserves, and this may be connected with the unchanged activity levels of GSHR. In contrast, high activity of SOD isoenzymes effectively scavenges superoxide anion, the activity of GSHPxs being unchanged, therefore hydrogen peroxide may not be sufficiently removed, and contribute to damage of cellular structures. However, the significantly increased activity of GST in old tapeworms points to effective removal of the toxic products of proteins and lipid oxidation, and various electrophilic substances. In the medial part of young tapeworms, increased TBARS levels and decreased GSH levels indicate intensification of oxidative stress. Decreased activities of SOD isoenzymes (twofold), CAT, GSHPxs and GSHR possibly contribute to this process. It seems that due to the relatively long distance from the intestinal wall there is
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no need to maintain a high activity of antioxidant enzymes. Only the activity of GST is increased. This probably provides sufficient protection of the cells of the medial part by detoxification of lipid peroxidation products and exogenous toxins. GST is efficient in this protective effect, since it can catalyze the conjugation of GSH with various electrophilic substances. During the reactions mediated by GST, the conjugates that are produced are actively secreted by the cells of tapeworms, with subsequent depletion of GSH. In old tapeworms we also observed reduced levels of oxidative stress indicated by the very low activity of MnSOD and unchanged activity of other enzymes and oxidative stress markers. The terminal part of young tapeworms was characterized by the highest level of both TBARS and GSH. This indicates intensification of oxidative stress, which is probably balanced by high concentrations of GSH. Antioxidant enzyme activities in young tapeworms were higher than in the medial but lower than in the anterior parts. In the terminal part of old tapeworms, we also found very high levels of lipid peroxidation, with simultaneous high levels of GSH, which may be related to the observed low activity of antioxidant enzymes (CuZnSOD, CAT, tot. GSHPx, GST) indicating diminution of antioxidant system in old tapeworms. In all parts of both, young and old tapeworms, we found very low activity of CAT. The observed values of CAT activity in our study are close to the limit of sensitivity of the analytical method. Our results are in line with observations of numerous authors that CAT activity is not found usually in most parasitic species (Callahan et al., 1988; HenkleDührsen and Kampkötter, 2001). Comparison of the activities of antioxidant enzymes in young and old tapeworms showed about 10-fold lower levels in all parts of old worms. Only the GST activity was higher, perhaps indicating diminution of antioxidant system in old tapeworms as a result of depletion and oxidative damage of relevant enzymes. However, we observed decreased level of TBARS in old tapeworms, which may suggest that the antioxidant system of old tapeworms has lower activity due to the weaker response of the immune system of the host. During infection, parasites are able to suppress defensive mechanisms of the host and adapt to oxidative stress conditions. After a long persistence of tapeworm in the host, this suppression cause the silencing of specific components of the host response to parasitic infection.
6. Conclusions The results reported in this paper indicate differences in the antioxidant systems of both the young and old forms of H. diminuta. Changes in the levels of oxidative stress markers and antioxidant enzymes activity indicate that tapeworms are exposed to high levels of ROS, a result of oxidative stress caused by the inflammatory state that is associated with parasitic infections. Due to the high activity of antioxidant enzymes, the anterior segments of young and old tapeworms are equipped with very effective antioxidant systems. To survive in the conditions of the inflamed intestinal state H. diminuta has adapted its metabolism to cope with the oxidative stress. This is mainly reflected in relatively high activity of the key antioxidant enzyme – SOD, particularly in the anterior segment of young tapeworms. Also the increased activity of total GSHPx points to an effective scavenging of organic peroxides, which are toxic derivatives of ROS. Relatively high concentrations of GSH in all segments of tapeworms also indicate good antioxidant defenses. On the other hand, decreased level of lipid peroxidation and high activity GST in old tapeworms indicate that old organisms effectively act against oxidative stress. This suggests finely tuned adaptation to the hostile environment.
Acknowledgments We are grateful to Professor Jerzy M. Behnke, School of Biology, University of Nottingham, UK for his kind help in the linguistic correction of the final version of this paper.
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