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Characterisation of Fasciola hepatica cytochrome c peroxidase as an enzyme with potential antioxidant activity in vitro
International Journal for Parasitology 29 (1999) 655±662
Characterisation of Fasciola hepatica cytochrome c peroxidase as an enzyme with potential antioxidant activity in vitro EÂlida G. Campos a, b, *, Marcelo Hermes-Lima b, James M. Smith a, Roger K. Prichard a a
Institute of Parasitology, McGill University, MacDonald Campus, 21,111 Lakeshore Road, Ste. Anne de Bellevue, Quebec, Canada H9X 3V9 b Departmento de Biologia Celular, Universidade de BrasõÂlia, BrasõÂlia, DF, CEP 70910-900, Brazil Received 14 December 1998; received in revised form 9 March 1999; accepted 9 March 1999
Abstract Cytochrome c peroxidase oxidises hydrogen peroxide using cytochrome c as the electron donor. This enzyme is found in yeast and bacteria and has been also described in the trematodes Fasciola hepatica and Schistosoma mansoni. Using partially puri®ed cytochrome c peroxidase samples from Fasciola hepatica we evaluated its role as an antioxidant enzyme via the investigation of its ability to protect against oxidative damage to deoxyribose in vitro. A system containing FeIII±EDTA plus ascorbate was used to generate reactive oxygen species superoxide radical, H2O2 as well as the hydroxyl radical. Fasciola hepatica cytochrome c peroxidase eectively protected deoxyribose against oxidative damage in the presence of its substrate cytochrome c. This protection was proportional to the amount of enzyme added and occurred only in the presence of cytochrome c. Due to the low speci®c activity of the ®nal partially puri®ed sample the eects of ascorbate and calcium chloride on cytochrome c peroxidase were investigated. The activity of the partially puri®ed enzyme was found to increase between 10 and 37% upon reduction with ascorbate. However, incubation of the partially puri®ed enzyme with 1 mM calcium chloride did not have any eect on enzyme activity. Our results showed that Fasciola hepatica CcP can protect deoxyribose from oxidative damage in vitro by blocking the formation of the highly toxic hydroxyl radical (OH). We suggest that the capacity of CcP to inhibit OH-formation, by eciently removing H2O2 from the in vitro oxidative system, may extend the biological role of CcP in response to oxidative stress in Fasciola hepatica. # 1999 Australian Society for Parasitology. Published by Elsevier Science Ltd. All rights reserved. Keywords: Cytochrome c peroxidase; Schistosoma mansoni; Fasciola hepatica; Reactive oxygen species; Antioxidant enzyme; Oxidative stress
* Corresponding author. Tel.: 55-61-307-2423; fax: 55-61-349-8411; e-mail: [email protected] 0020-7519/$20.00 # 1999 Australian Society for Parasitology. Published by Elsevier Science Ltd. All rights reserved. PII: S 0 0 2 0 - 7 5 1 9 ( 9 9 ) 0 0 0 3 0 - 2
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1. Introduction Cytochrome c peroxidase (CcP; EC 1.11.1.5) is a hemeprotein found in yeast (Saccharomyces cerevisiae) and bacteria, in some lower eukaryotes, such as the trematodes Fasciola hepatica, and Schistosoma mansoni [1±3]. Cytochrome c peroxidase oxidises H2O2 using cytochrome c as the electron donor. This enzyme is present in the intermembrane space of S. cerevisiae mitochondria [4]. Yeast CcP has a characteristic property of undergoing the so-called `induced conversion' which is the formation of the holoenzyme (by attachment of the heme moiety) when anaerobic yeast adapts to oxygen [5]. It has been shown that, under physiological conditions, yeast CcP utilises 53±55% of the H2O2 generated at the level of the mitochondrial respiratory chain, thus preventing H2O2 leakage to the cytosol [6]. Studies of the mechanism of reaction and protein structure of CcP, both from baker's yeast and bacteria, have generated an abundance of information about this enzyme [4, 7]. However, CcP appears to be absent from vertebrates and little is known about its physiological role in the lower multicellular organisms in which it has been described. Cytochrome c peroxidase activity is present in F. hepatica [2] but its function and importance for the parasite remain to be determined. Fasciola hepatica is a trematode parasite which infects cattle, sheep, goats, and other mammals including humans. This parasite utilises oxygen when available [8]. In addition, it has been shown that the respiration of the whole adult worm is dependent on the prevailing oxygen tensions [9]. Hydrogen peroxide is formed as an end product of respiration in F. hepatica [8] and worms rely on the activity of peroxidases for removal of the formed H2O2. Catalase, which is an ubiquitous enzyme involved in the removal of H2O2 in cells has not been detected in F. hepatica and S. mansoni [2, 10]. However, the presence of abundant CcP activity may compensate for the lack of catalase in these organisms. Previous puri®cation studies with CcP from F. hepatica and S. mansoni indicated that the trematode enzyme is a heme protein composed of two identical subunits of approximately 31 KDa
(Campos EG, Ph.D. Thesis, McGill University, 1996). In the present article, using the partially puri®ed enzyme, the antioxidant properties of CcP from F. hepatica were tested by determining whether the enzyme could inhibit damage to deoxyribose by reactive oxygen species (ROS), in vitro. Hydrogen peroxide participates in reactions where iron or iron±EDTA complexes function as catalysts in the generation of deleterious oxygen species [11, 12] which can cause damage to cell membrane components, enzymes, DNA and sugar molecules [13, 14]. It has been reported that oxidative damage to the carbohydrate deoxyribose produces thiobarbituric acid reactive substances (TBA-RS) with ¯uorescence identical to malondialdehyde [15]. Our experimental approach in this study involved the quanti®cation of TBA-RS generated by the oxidative damage to deoxyribose. The FeIII±EDTA-ascorbate system was used to generate ROS [16]. 2. Materials and methods 2.1. Fasciola hepatica adult worms Adult worms were obtained from the liver and bile ducts of infected sheep 7 weeks p.i. The worms were collected in HeÂdon±Fleig solution containing no antibiotics according to Dawes [17]. After collection, the worms were incubated for 3±5 h at 378C in fresh HeÂdon±Fleig solution in order to allow the gut contents to be excreted by the worms. After this period, the worms appeared normal, moved actively and had a homogeneous pink colour. They were then washed in fresh medium, frozen in liquid nitrogen and stored at ÿ708C until needed. 2.2. Isolation of Fasciola hepatica CcP Partial puri®cation of F. hepatica CcP was performed as follows: worms were homogenised in 50 mM sodium acetate buer, pH 6.0 containing 1.25 mM EDTA, 1 mM PMSF, 1 mM benzamidine, pepstatin A (1 mg mlÿ1) and leupeptin (1 mg mlÿ1). The crude homogenate was sonicated
E.G. Campos et al./International Journal for Parasitology 29 (1999) 655±662
using a Vibra Cell2 sonicator. After sonication, the sample was centrifuged at 110 000 gÿ1 for 1 h and the supernatant collected and used for further enzyme puri®cation. A 40% solution of polyethylene glycol 8000 g was added to the supernatant to give a ®nal concentration of 2% and the sample was left rotating in a cold room at 48C for 1 h. After incubation, the sample was centrifuged at 8000 g for 15 min and the supernatant containing CcP activity was collected and applied to a Q-Sepharose ion-exchange column (Flast ¯ow anion exchanger, Q-1126, Sigma) in sodium acetate buer, 0.05 M (pH 6.0). Bound proteins were eluted with a continuous gradient of 0±1 M KCl in acetate buer. The fractions from the Q-Sepharose which had CcP activity were combined, concentrated to a volume of 500 ml using a Centriprep-10 concentrator (Amicon Inc.) and applied directly to a pre-packed gel ®ltration column (Sephacryl S-100-Pharmacia) equilibrated with 50 mM sodium acetate buer (pH 5.0) containing 1 mM PMSF and 0.15 M NaCl. The sample (500 ml) applied to the gel ®ltration column was eluted using a ¯ow rate of 0.1 ml minÿ1. The fractions in which CcP activity was detected were pooled and used for the experiments described in this study. Protein samples from F. hepatica were fractionated by PAGE using 12% acrylamide pre-casted gels (BioRad) and the buer system of Laemmli [18]. The heme detection method was based on the peroxidase activity of heme using 3,3 0 ,5,5 0 -tetramethylbenzidine as an oxidizable substrate [19]. 2.3. Measurement of CcP activity Enzyme activity was monitored by following the oxidation of cytochrome c at 550 nm at 258C. Fully reduced cytochrome c was prepared as described by Yonetani and Ray [20]. The reaction mixture contained (1 ml): 50 mM Na acetate buer (pH 6.0), 35 mM cytochrome c, 70 mM H2O2 and sample. Control reactions consisted of reaction mixture in the absence of either sample or H2O2. A solution of approximately 9.2 mM H2O2 was obtained by a 980-fold dilution with water of a 30% H2O2 solution. The concen-
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tration of the stock solution was calculated each day from the absorbance at 230 nm (E = 81 cm ÿ 1 M ÿ 1). Protein concentration was measured by the method of Bradford [21] using bovine serum albumine as standard. 2.4. Ascorbate and Ca2 + addition to cytochrome c peroxidase activity Sodium ascorbate (0.1 M) and calcium chloride (0.1 M) were prepared in 50 mM sodium acetate buer, pH 6.0. The fractions having CcP activity which were collected from the gel ®ltration column were used to measure the eects of ascorbate and calcium on enzyme activity. Samples of 100 ml (8±25 mg protein mlÿ1) were incubated at room temperature with 2 mM sodium ascorbate. Aliquots of the samples (20 ml) were assayed for activity after 30 and 60 min of incubation. In addition, after 45 min of incubation with 2 mM ascorbate, 50 ml of each sample were transferred to a new tube to which 1 mM of calcium chloride was added. The activity of this sample was measured after 15 min in the presence of 1 mM calcium in the reaction mixture. 2.5. The FeIII±EDTA-ascorbate system The assay of deoxyribose degradation used in this study was based on that described by Hermes-Lima et al. [16] with a few modi®cations. The total volume of the reaction mixture used was 0.5 ml. The mixture contained 20 mM potassium phosphate buer, pH 7.0, 10 mM deoxyribose, 100 mM EDTA, 10 mM FeCl3 and 2 mM sodium ascorbate. When reduced cytochrome c was included in the reaction, the concentration used was 35 mM. In the reactions containing the partially isolated CcP from F. hepatica, 20 ml of enzyme solution were used, corresponding to an average of 0.5 mg of protein. The concentration of KCN and sodium azide used was 2 mM. The reagents were added in the following order: water, deoxyribose, EDTA, FeIII, cytochrome c, ascorbate and ®nally the enzyme. The following controls were used: (1) the reaction mixture without the sodium ascorbate, (2) with-
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E.G. Campos et al./International Journal for Parasitology 29 (1999) 655±662
out the cytochrome c, and (3) without the enzyme. Blanks contained all reactants, including ascorbate, but were stopped at time zero. After all reagents had been added to the reaction mixture, the tubes were transferred to a 378C shaking water bath and allowed to incubate for predetermined periods of time (see Section 3). TBA reagent (1%) was prepared in 50 mM NaOH and 0.5 ml of 10 mM butylated hydroxytoluene (BHT, Sigma) was added to each 50 ml TBA solution. Following incubation, 0.5 ml of TBA reagent was added to all tubes and samples were boiled for 10 min. When samples reached room temperature, the proteins were extracted by adding 1.5 ml butanol and vortexing vigorously for 40 s. Tubes were centrifuged at 1900 g for 5 min. The sample separated into three distinct layers, the top organic phase, the middle protein layer and the bottom aqueous layer. The top layer was transferred to a cuvette and scanned between wavelengths 400 and 600 nm. The values for the absorbance peak at 535 nm were then recorded. 2.6. Analysis of data Analysis of variance (ANOVA) followed by Dunnett's test was used to test the eects of ascorbate and calcium on CcP activity and Student's t-test (Figs. 3±5) were used for statistical analysis and signi®cance was taken at P < 0.05 (95% CI). Bars in the results represent S.E.M.
3. Results The enzymatic oxidation of horse heart cytochrome c by partially puri®ed CcP from F. hepatica was measured as described in Section 2. The speci®c activity of the partially puri®ed sample was 16 U mgÿ1 protein, where units (U) were expressed as mmol of horse heart ferrocytochrome c oxidised per min per ml of reaction mixture. The speci®c activity in crude extracts varied between 0.7 and 2 U mgÿ1. The average recovery of F. hepatica CcP activity was 2.5%.
Fig. 1. SDS±PAGE of the partially puri®ed preparation of cytochrome c peroxidase from Fasciola hepatica. (A) Sample collected from Q-Sepharose column and concentrated by Centricon 10; (B) Sample collected from Sephacryl S-100 gel ®ltration column. After the run, the 12% polyacrylamide gel was ®rst stained by the heme detection method followed by Coomassie blue stain. The position of the protein band which stained for heme is labelled with the asterisk (*). A faint heme staining band was also observed at the position labelled by (* 0 ). The numbers refer to molecular mass in kDa as determined from marker proteins. S = top of the separating gel.
The partially puri®ed sample collected from the gel ®ltration column showed, upon Coomassie blue staining, four major protein bands and some minor contaminants (Fig. 1). One of the major bands stained strongly for heme, indicating that it is a cytochrome. Fig. 2 shows the dierence between the oxidation of reduced cytochrome c by H2O2 and its enzymatic oxidation catalysed by partially puri®ed CcP. The rate of Cc oxidation in the presence of CcP was 5.3 times higher than in its absence. Puri®ed CcPs from Paracoccus denitri®cans and Pseudomonas aeruginosa have been shown to be peroxidatically inactive towards cytochrome c, unless they are partially reduced before the peroxidation reaction is measured [22, 23]. To investigate if reduction of partially puri®ed CcP from F. hepatica would increase its speci®c activity we treated the sample with ascorbate prior to
E.G. Campos et al./International Journal for Parasitology 29 (1999) 655±662
measuring enzyme activity. The results showed that when CcP was incubated with ascorbate for 30 and 60 min at room temperature, CcP activity increased by 10±37%. In this present study, incubation of F. hepatica CcP with ascorbate plus calcium did not cause a statistically signi®cant increase in enzyme activity in relation to ascorbate alone (4.612 0.2 versus 4.99 2 0.2 nmol minÿ1), although it was statistically dierent from the control without ascorbate and calcium (3.86 20.3 mmol minÿ110 ÿ 3). The increase in activity of samples treated with ascorbate plus calcium, compared with samples without ascorbate or calcium ranged between 18 and 53%. CcP samples partially puri®ed from F. hepatica were used to test the capacity of cytochrome c peroxidase to act as an antioxidant in vitro. The experiments were performed with three independently partially puri®ed preparations of F. hepatica CcP. Figure 3a shows a time course study of the eect, caused by the presence of CcP, on the formation of TBA-RS due to the oxidative
Fig. 2. Oxidation of cytochrome c by partially puri®ed CcP from Fasciola hepatica (enzymatic reaction) and its reaction with H2O2 (non-enzymatic reaction). Oxidation of cytochrome c was measured by the decrease in absorbance at 550 nm. The reaction mixture contained 0.05 M sodium acetate buer pH 6.0, 70 mM of H2O2 and cytochrome c (35 mM) or cytochrome c plus CcP (0.5 mg protein). The rates for the enzymatic and non-enzymatic reactions were 0.016 and 0.003 mmol minÿ1, respectively.
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Fig. 3. Oxidative damage of deoxyribose measured by the formation of TBA-RS and protection by CcP. The reaction was incubated at 37 8C for dierent time intervals before it was stopped by the addition of the TBA reagent. The reaction mixture contained potassium phosphate buer (20 mM, pH 7.0), deoxyribose (10 mM), EDTA (100 mM), FeCl3 (10 mM), ascorbic acid (2mM) and reduced horse heart cytochrome c (Cc, 35 mM) in 0.5 ml of reaction mixture. (A) Twenty ml of the sample (0.5 mg protein) containing CcP activity, which was collected from the Sephacryl S-100 column, were used together with cytochrome c (CcP + Cc). The control reaction had neither cytochrome c peroxidase nor cytochrome c (No CcP or Cc). There was a signi®cant reduction in TBA-RS formation at 1 h (P = 0.0068, CI 95%) and at 2 h (P = 0.0419, CI 95%). (B) Percentage inhibition of TBA-RS in relation to the control reaction and the eect of KCN (2 mM) present in the reaction mixture after 2 h incubation. Number of determinations, n = 3. Where error bars do not show they are smaller than the dots.
degradation of deoxyribose. There was a linear relationship between the incubation time and the level of TBA-RS formed. Cytochrome c peroxidase plus horse heart cytochrome c caused a decrease in TBA-RS formation when compared with the control reaction. The inhibition of TBARS formation is shown in Fig. 3b. Cytochrome c alone inhibited TBA-RS formation by approximately 10%, while cytochrome c plus CcP inhibited TBA-RS by about 25%. The addition of 2 mM KCN reversed the protection given by cytochrome c peroxidase after a 2 h incubation (Fig. 3b). Potassium cyanide present in the reaction mixture completely blocked the antioxidant eect of CcP plus cytochrome c. We also tested the eect of sodium azide and observed that it inhibited the formation of TBA-RS (not shown).
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Fig. 4. Decrease in TBA-RS formation as a function of the amount of CcP added to the FeIII±EDTA-ascorbate system. The formation of TBA-RS was measured using increasing amounts of CcP (ml of enzyme) in the presence and absence of the enzyme's substrate, cytochrome c (Cc, 35 mM). Incubation time was 2 h. There was a signi®cant reduction in TBA-RS formation with increase in enzyme concentration in the presence of the substrate cytochrome c (P = 0.0029) but not in the absence of substrate. Data points with the same letter are not signi®cantly dierent.
The inhibitory eect of cytochrome c peroxidase on deoxyribose damage was proportional to the amount of enzyme added (Fig. 4). This eect was observed only when the substrate (cytochrome c) was included in the reaction mixture; a strong indication that the inhibition of TBA-RS formation is due to the enzymatic removal of H2O2 from the reaction mixture and not to heme present in the enzyme added. Figure 5 shows the eect of increasing amounts of FeCl3 on TBARS formation. The protection given by F. hepatica CcP occurred at 10 mM of FeCl3, but not when FeCl3 concentration was increased to 20 and 30 mM. 4. Discussion To investigate the possible causes of the low recovery of enzyme activity during puri®cation, ascorbate was used to reduce the enzyme. For the bacterial di-heme cytochrome c peroxidases, activity seems to be modulated by the oxidation states of the hemes. For example, cytochrome c
peroxidase from P. aeruginosa and P. denitri®cans require reduction of the high potential heme before catalysis can occur [22, 23]. On the other hand, CcP from Nitrosomonas europaea is active in the fully oxidised and partially reduced states [24]. Reduction of the F. hepatica CcP with ascorbate increased the enzyme activity indicating that the oxidative state of the enzyme is important for activity. The increase in F. hepatica CcP activity upon ascorbate reduction was measured in the absence of an electron-transfer mediator in the incubation medium and represents the minimum eect of ascorbate. The eect of calcium on F. hepatica CcP activity was also investigated. Calcium ions are important for horseradish peroxidase activity [25] and seems to be essential for the activation of Paracoccus cytochrome c peroxidase [23]. Yeast cytochrome c peroxidase, in contrast, contains no
Fig. 5. Addition of increasing amounts of FeCl3 to the FeIII± EDTA-ascorbate system increased the formation of TBA-RS and abolished the protection aorded by CcP. Cytochrome c (21 mM) and Cc (21 mM) plus CcP (60 ml) were used. All other reagents in the reaction mixture were the same as described in Fig. 3. The incubation time was 2 h. (A) TBARS formation measured by the absorbance peak at 535 nm. The reduction in TBA-RS formation in the presence of CcP + Cc at a concentration of 10 mM FeCl3 is signi®catively dierent from Cc alone (P = 0.008). (B) Percentage inhibition in relation to the control reaction. Empty bars (CcP + Cc), ®lled bars (Cc alone). Number of determinations, n = 3.
E.G. Campos et al./International Journal for Parasitology 29 (1999) 655±662
calcium [26]. In this study calcium seemed not to be involved in F. hepatica CcP activation. In a study of the kinetics of the oxidative deoxyribose degradation reaction catalysed by FeIII±EDTA plus ascorbate, an increase in the amount of FeCl3 added to the reaction (up to 15 mM) promoted a saturable rise in the yields of deoxyribose degradation in the presence of EDTA and ascorbate [16]. This saturation was obtained using 3 mM of ascorbate and 15 mM of deoxyribose compared with 2 and 10 mM, respectively, used in the present study. Based on the results presented in Fig. 5 it can be postulated that the antioxidant activity of CcP is overwhelmed by high levels of H2O2 formation, and consequently hydroxyl radicals, reached at 20 and 30 mM FeCl3. Peroxidases detoxify H2O2, which can damage multiple cellular components. Hydrogen peroxide eects on biological systems have been reviewed by Cochrane [27]. The glycolytic pathway as well as lactate production, for example, were inhibited when P388D1 cells where exposed to H2O2 in vitro [27]. DNA damage has been shown in several cell types which had been exposed to H2O2. The ability of H2O2 to induce DNA strand breaks has been shown to require the presence of FeII, indicating that the mechanism of damage involved the generation of OH from H2O2, through the Fenton Reaction [28]. Thus, H2O2 can penetrate cells and diuse through the cellular compartments, including the nuclei, to cause DNA damage. Therefore, the presence of peroxidases make the cells resistant to DNA damage caused by H2O2. A speci®c physiological function for CcP in F. hepatica has still to be determined, but is most probably linked to the nature and functioning of the intracellular sources of H2O2 and its H2O2 scavenging activity. It has been demonstrated that H2O2 is produced by the mitochondria of adult F. hepatica [8]. Hydrogen peroxide has been considered a normal metabolite in cells, present at steady-state concentrations in the range of 10 ÿ 8 to 10 ÿ 9 M [29]. It can also be formed from superoxide anions which are produced by phagocytic cells [30] and may be present at much higher concentrations at sites of in¯ammation.
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Few studies have dealt with the production of H2O2 by the host immune cells during infections with F. hepatica and the role it may play in resistance or susceptibility to fascioliasis. Smith et al. [31] have found higher production of free radicals by peritoneal leukocytes from infected nonpermissive hosts (rats) compared with leukocytes from infected permissive hosts (mice). On the other hand, BaeÂza et al. [32] found that ES products of F. hepatica caused a decrease in the production of free radicals by bovine polymorphonuclear leukocytes. Thus, it has still to be clari®ed if F. hepatica can inhibit the respiratory burst of host immune cells or if it can detoxify ROS which are formed, avoiding oxidative damage. Any role for cytochrome c peroxidase in protecting F. hepatica or other trematodes, such as S. mansoni [3, 33] from host oxidative attack remains to be determined. Cytochrome c peroxidase might play a role in protection against host immune oxidative attack.
Acknowledgements We acknowledge the contribution of the CIDA/McGill Fellowship Program and the Brazilian Research Council (CNPq/PRONEX), which supported E.G.C. and M.H.L. We are grateful to Christiane Trudeau for her technical assistance and animal care. This project was supported, in part, by a MRC grant to R.K.P. and J.M.S. Research at the Institute of Parasitology is supported by Fonds FCAR pour la Formation des Chercheurs et l'Aide aÁ la Recherche.
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Characterisation of Fasciola hepatica cytochrome c peroxidase as an enzyme with potential antioxidant activity in vitro EÂlida G. Campos a, b, *, Marcelo Hermes-Lima b, James M. Smith a, Roger K. Prichard a a
Institute of Parasitology, McGill University, MacDonald Campus, 21,111 Lakeshore Road, Ste. Anne de Bellevue, Quebec, Canada H9X 3V9 b Departmento de Biologia Celular, Universidade de BrasõÂlia, BrasõÂlia, DF, CEP 70910-900, Brazil Received 14 December 1998; received in revised form 9 March 1999; accepted 9 March 1999
Abstract Cytochrome c peroxidase oxidises hydrogen peroxide using cytochrome c as the electron donor. This enzyme is found in yeast and bacteria and has been also described in the trematodes Fasciola hepatica and Schistosoma mansoni. Using partially puri®ed cytochrome c peroxidase samples from Fasciola hepatica we evaluated its role as an antioxidant enzyme via the investigation of its ability to protect against oxidative damage to deoxyribose in vitro. A system containing FeIII±EDTA plus ascorbate was used to generate reactive oxygen species superoxide radical, H2O2 as well as the hydroxyl radical. Fasciola hepatica cytochrome c peroxidase eectively protected deoxyribose against oxidative damage in the presence of its substrate cytochrome c. This protection was proportional to the amount of enzyme added and occurred only in the presence of cytochrome c. Due to the low speci®c activity of the ®nal partially puri®ed sample the eects of ascorbate and calcium chloride on cytochrome c peroxidase were investigated. The activity of the partially puri®ed enzyme was found to increase between 10 and 37% upon reduction with ascorbate. However, incubation of the partially puri®ed enzyme with 1 mM calcium chloride did not have any eect on enzyme activity. Our results showed that Fasciola hepatica CcP can protect deoxyribose from oxidative damage in vitro by blocking the formation of the highly toxic hydroxyl radical (OH). We suggest that the capacity of CcP to inhibit OH-formation, by eciently removing H2O2 from the in vitro oxidative system, may extend the biological role of CcP in response to oxidative stress in Fasciola hepatica. # 1999 Australian Society for Parasitology. Published by Elsevier Science Ltd. All rights reserved. Keywords: Cytochrome c peroxidase; Schistosoma mansoni; Fasciola hepatica; Reactive oxygen species; Antioxidant enzyme; Oxidative stress
* Corresponding author. Tel.: 55-61-307-2423; fax: 55-61-349-8411; e-mail: [email protected] 0020-7519/$20.00 # 1999 Australian Society for Parasitology. Published by Elsevier Science Ltd. All rights reserved. PII: S 0 0 2 0 - 7 5 1 9 ( 9 9 ) 0 0 0 3 0 - 2
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1. Introduction Cytochrome c peroxidase (CcP; EC 1.11.1.5) is a hemeprotein found in yeast (Saccharomyces cerevisiae) and bacteria, in some lower eukaryotes, such as the trematodes Fasciola hepatica, and Schistosoma mansoni [1±3]. Cytochrome c peroxidase oxidises H2O2 using cytochrome c as the electron donor. This enzyme is present in the intermembrane space of S. cerevisiae mitochondria [4]. Yeast CcP has a characteristic property of undergoing the so-called `induced conversion' which is the formation of the holoenzyme (by attachment of the heme moiety) when anaerobic yeast adapts to oxygen [5]. It has been shown that, under physiological conditions, yeast CcP utilises 53±55% of the H2O2 generated at the level of the mitochondrial respiratory chain, thus preventing H2O2 leakage to the cytosol [6]. Studies of the mechanism of reaction and protein structure of CcP, both from baker's yeast and bacteria, have generated an abundance of information about this enzyme [4, 7]. However, CcP appears to be absent from vertebrates and little is known about its physiological role in the lower multicellular organisms in which it has been described. Cytochrome c peroxidase activity is present in F. hepatica [2] but its function and importance for the parasite remain to be determined. Fasciola hepatica is a trematode parasite which infects cattle, sheep, goats, and other mammals including humans. This parasite utilises oxygen when available [8]. In addition, it has been shown that the respiration of the whole adult worm is dependent on the prevailing oxygen tensions [9]. Hydrogen peroxide is formed as an end product of respiration in F. hepatica [8] and worms rely on the activity of peroxidases for removal of the formed H2O2. Catalase, which is an ubiquitous enzyme involved in the removal of H2O2 in cells has not been detected in F. hepatica and S. mansoni [2, 10]. However, the presence of abundant CcP activity may compensate for the lack of catalase in these organisms. Previous puri®cation studies with CcP from F. hepatica and S. mansoni indicated that the trematode enzyme is a heme protein composed of two identical subunits of approximately 31 KDa
(Campos EG, Ph.D. Thesis, McGill University, 1996). In the present article, using the partially puri®ed enzyme, the antioxidant properties of CcP from F. hepatica were tested by determining whether the enzyme could inhibit damage to deoxyribose by reactive oxygen species (ROS), in vitro. Hydrogen peroxide participates in reactions where iron or iron±EDTA complexes function as catalysts in the generation of deleterious oxygen species [11, 12] which can cause damage to cell membrane components, enzymes, DNA and sugar molecules [13, 14]. It has been reported that oxidative damage to the carbohydrate deoxyribose produces thiobarbituric acid reactive substances (TBA-RS) with ¯uorescence identical to malondialdehyde [15]. Our experimental approach in this study involved the quanti®cation of TBA-RS generated by the oxidative damage to deoxyribose. The FeIII±EDTA-ascorbate system was used to generate ROS [16]. 2. Materials and methods 2.1. Fasciola hepatica adult worms Adult worms were obtained from the liver and bile ducts of infected sheep 7 weeks p.i. The worms were collected in HeÂdon±Fleig solution containing no antibiotics according to Dawes [17]. After collection, the worms were incubated for 3±5 h at 378C in fresh HeÂdon±Fleig solution in order to allow the gut contents to be excreted by the worms. After this period, the worms appeared normal, moved actively and had a homogeneous pink colour. They were then washed in fresh medium, frozen in liquid nitrogen and stored at ÿ708C until needed. 2.2. Isolation of Fasciola hepatica CcP Partial puri®cation of F. hepatica CcP was performed as follows: worms were homogenised in 50 mM sodium acetate buer, pH 6.0 containing 1.25 mM EDTA, 1 mM PMSF, 1 mM benzamidine, pepstatin A (1 mg mlÿ1) and leupeptin (1 mg mlÿ1). The crude homogenate was sonicated
E.G. Campos et al./International Journal for Parasitology 29 (1999) 655±662
using a Vibra Cell2 sonicator. After sonication, the sample was centrifuged at 110 000 gÿ1 for 1 h and the supernatant collected and used for further enzyme puri®cation. A 40% solution of polyethylene glycol 8000 g was added to the supernatant to give a ®nal concentration of 2% and the sample was left rotating in a cold room at 48C for 1 h. After incubation, the sample was centrifuged at 8000 g for 15 min and the supernatant containing CcP activity was collected and applied to a Q-Sepharose ion-exchange column (Flast ¯ow anion exchanger, Q-1126, Sigma) in sodium acetate buer, 0.05 M (pH 6.0). Bound proteins were eluted with a continuous gradient of 0±1 M KCl in acetate buer. The fractions from the Q-Sepharose which had CcP activity were combined, concentrated to a volume of 500 ml using a Centriprep-10 concentrator (Amicon Inc.) and applied directly to a pre-packed gel ®ltration column (Sephacryl S-100-Pharmacia) equilibrated with 50 mM sodium acetate buer (pH 5.0) containing 1 mM PMSF and 0.15 M NaCl. The sample (500 ml) applied to the gel ®ltration column was eluted using a ¯ow rate of 0.1 ml minÿ1. The fractions in which CcP activity was detected were pooled and used for the experiments described in this study. Protein samples from F. hepatica were fractionated by PAGE using 12% acrylamide pre-casted gels (BioRad) and the buer system of Laemmli [18]. The heme detection method was based on the peroxidase activity of heme using 3,3 0 ,5,5 0 -tetramethylbenzidine as an oxidizable substrate [19]. 2.3. Measurement of CcP activity Enzyme activity was monitored by following the oxidation of cytochrome c at 550 nm at 258C. Fully reduced cytochrome c was prepared as described by Yonetani and Ray [20]. The reaction mixture contained (1 ml): 50 mM Na acetate buer (pH 6.0), 35 mM cytochrome c, 70 mM H2O2 and sample. Control reactions consisted of reaction mixture in the absence of either sample or H2O2. A solution of approximately 9.2 mM H2O2 was obtained by a 980-fold dilution with water of a 30% H2O2 solution. The concen-
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tration of the stock solution was calculated each day from the absorbance at 230 nm (E = 81 cm ÿ 1 M ÿ 1). Protein concentration was measured by the method of Bradford [21] using bovine serum albumine as standard. 2.4. Ascorbate and Ca2 + addition to cytochrome c peroxidase activity Sodium ascorbate (0.1 M) and calcium chloride (0.1 M) were prepared in 50 mM sodium acetate buer, pH 6.0. The fractions having CcP activity which were collected from the gel ®ltration column were used to measure the eects of ascorbate and calcium on enzyme activity. Samples of 100 ml (8±25 mg protein mlÿ1) were incubated at room temperature with 2 mM sodium ascorbate. Aliquots of the samples (20 ml) were assayed for activity after 30 and 60 min of incubation. In addition, after 45 min of incubation with 2 mM ascorbate, 50 ml of each sample were transferred to a new tube to which 1 mM of calcium chloride was added. The activity of this sample was measured after 15 min in the presence of 1 mM calcium in the reaction mixture. 2.5. The FeIII±EDTA-ascorbate system The assay of deoxyribose degradation used in this study was based on that described by Hermes-Lima et al. [16] with a few modi®cations. The total volume of the reaction mixture used was 0.5 ml. The mixture contained 20 mM potassium phosphate buer, pH 7.0, 10 mM deoxyribose, 100 mM EDTA, 10 mM FeCl3 and 2 mM sodium ascorbate. When reduced cytochrome c was included in the reaction, the concentration used was 35 mM. In the reactions containing the partially isolated CcP from F. hepatica, 20 ml of enzyme solution were used, corresponding to an average of 0.5 mg of protein. The concentration of KCN and sodium azide used was 2 mM. The reagents were added in the following order: water, deoxyribose, EDTA, FeIII, cytochrome c, ascorbate and ®nally the enzyme. The following controls were used: (1) the reaction mixture without the sodium ascorbate, (2) with-
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out the cytochrome c, and (3) without the enzyme. Blanks contained all reactants, including ascorbate, but were stopped at time zero. After all reagents had been added to the reaction mixture, the tubes were transferred to a 378C shaking water bath and allowed to incubate for predetermined periods of time (see Section 3). TBA reagent (1%) was prepared in 50 mM NaOH and 0.5 ml of 10 mM butylated hydroxytoluene (BHT, Sigma) was added to each 50 ml TBA solution. Following incubation, 0.5 ml of TBA reagent was added to all tubes and samples were boiled for 10 min. When samples reached room temperature, the proteins were extracted by adding 1.5 ml butanol and vortexing vigorously for 40 s. Tubes were centrifuged at 1900 g for 5 min. The sample separated into three distinct layers, the top organic phase, the middle protein layer and the bottom aqueous layer. The top layer was transferred to a cuvette and scanned between wavelengths 400 and 600 nm. The values for the absorbance peak at 535 nm were then recorded. 2.6. Analysis of data Analysis of variance (ANOVA) followed by Dunnett's test was used to test the eects of ascorbate and calcium on CcP activity and Student's t-test (Figs. 3±5) were used for statistical analysis and signi®cance was taken at P < 0.05 (95% CI). Bars in the results represent S.E.M.
3. Results The enzymatic oxidation of horse heart cytochrome c by partially puri®ed CcP from F. hepatica was measured as described in Section 2. The speci®c activity of the partially puri®ed sample was 16 U mgÿ1 protein, where units (U) were expressed as mmol of horse heart ferrocytochrome c oxidised per min per ml of reaction mixture. The speci®c activity in crude extracts varied between 0.7 and 2 U mgÿ1. The average recovery of F. hepatica CcP activity was 2.5%.
Fig. 1. SDS±PAGE of the partially puri®ed preparation of cytochrome c peroxidase from Fasciola hepatica. (A) Sample collected from Q-Sepharose column and concentrated by Centricon 10; (B) Sample collected from Sephacryl S-100 gel ®ltration column. After the run, the 12% polyacrylamide gel was ®rst stained by the heme detection method followed by Coomassie blue stain. The position of the protein band which stained for heme is labelled with the asterisk (*). A faint heme staining band was also observed at the position labelled by (* 0 ). The numbers refer to molecular mass in kDa as determined from marker proteins. S = top of the separating gel.
The partially puri®ed sample collected from the gel ®ltration column showed, upon Coomassie blue staining, four major protein bands and some minor contaminants (Fig. 1). One of the major bands stained strongly for heme, indicating that it is a cytochrome. Fig. 2 shows the dierence between the oxidation of reduced cytochrome c by H2O2 and its enzymatic oxidation catalysed by partially puri®ed CcP. The rate of Cc oxidation in the presence of CcP was 5.3 times higher than in its absence. Puri®ed CcPs from Paracoccus denitri®cans and Pseudomonas aeruginosa have been shown to be peroxidatically inactive towards cytochrome c, unless they are partially reduced before the peroxidation reaction is measured [22, 23]. To investigate if reduction of partially puri®ed CcP from F. hepatica would increase its speci®c activity we treated the sample with ascorbate prior to
E.G. Campos et al./International Journal for Parasitology 29 (1999) 655±662
measuring enzyme activity. The results showed that when CcP was incubated with ascorbate for 30 and 60 min at room temperature, CcP activity increased by 10±37%. In this present study, incubation of F. hepatica CcP with ascorbate plus calcium did not cause a statistically signi®cant increase in enzyme activity in relation to ascorbate alone (4.612 0.2 versus 4.99 2 0.2 nmol minÿ1), although it was statistically dierent from the control without ascorbate and calcium (3.86 20.3 mmol minÿ110 ÿ 3). The increase in activity of samples treated with ascorbate plus calcium, compared with samples without ascorbate or calcium ranged between 18 and 53%. CcP samples partially puri®ed from F. hepatica were used to test the capacity of cytochrome c peroxidase to act as an antioxidant in vitro. The experiments were performed with three independently partially puri®ed preparations of F. hepatica CcP. Figure 3a shows a time course study of the eect, caused by the presence of CcP, on the formation of TBA-RS due to the oxidative
Fig. 2. Oxidation of cytochrome c by partially puri®ed CcP from Fasciola hepatica (enzymatic reaction) and its reaction with H2O2 (non-enzymatic reaction). Oxidation of cytochrome c was measured by the decrease in absorbance at 550 nm. The reaction mixture contained 0.05 M sodium acetate buer pH 6.0, 70 mM of H2O2 and cytochrome c (35 mM) or cytochrome c plus CcP (0.5 mg protein). The rates for the enzymatic and non-enzymatic reactions were 0.016 and 0.003 mmol minÿ1, respectively.
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Fig. 3. Oxidative damage of deoxyribose measured by the formation of TBA-RS and protection by CcP. The reaction was incubated at 37 8C for dierent time intervals before it was stopped by the addition of the TBA reagent. The reaction mixture contained potassium phosphate buer (20 mM, pH 7.0), deoxyribose (10 mM), EDTA (100 mM), FeCl3 (10 mM), ascorbic acid (2mM) and reduced horse heart cytochrome c (Cc, 35 mM) in 0.5 ml of reaction mixture. (A) Twenty ml of the sample (0.5 mg protein) containing CcP activity, which was collected from the Sephacryl S-100 column, were used together with cytochrome c (CcP + Cc). The control reaction had neither cytochrome c peroxidase nor cytochrome c (No CcP or Cc). There was a signi®cant reduction in TBA-RS formation at 1 h (P = 0.0068, CI 95%) and at 2 h (P = 0.0419, CI 95%). (B) Percentage inhibition of TBA-RS in relation to the control reaction and the eect of KCN (2 mM) present in the reaction mixture after 2 h incubation. Number of determinations, n = 3. Where error bars do not show they are smaller than the dots.
degradation of deoxyribose. There was a linear relationship between the incubation time and the level of TBA-RS formed. Cytochrome c peroxidase plus horse heart cytochrome c caused a decrease in TBA-RS formation when compared with the control reaction. The inhibition of TBARS formation is shown in Fig. 3b. Cytochrome c alone inhibited TBA-RS formation by approximately 10%, while cytochrome c plus CcP inhibited TBA-RS by about 25%. The addition of 2 mM KCN reversed the protection given by cytochrome c peroxidase after a 2 h incubation (Fig. 3b). Potassium cyanide present in the reaction mixture completely blocked the antioxidant eect of CcP plus cytochrome c. We also tested the eect of sodium azide and observed that it inhibited the formation of TBA-RS (not shown).
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Fig. 4. Decrease in TBA-RS formation as a function of the amount of CcP added to the FeIII±EDTA-ascorbate system. The formation of TBA-RS was measured using increasing amounts of CcP (ml of enzyme) in the presence and absence of the enzyme's substrate, cytochrome c (Cc, 35 mM). Incubation time was 2 h. There was a signi®cant reduction in TBA-RS formation with increase in enzyme concentration in the presence of the substrate cytochrome c (P = 0.0029) but not in the absence of substrate. Data points with the same letter are not signi®cantly dierent.
The inhibitory eect of cytochrome c peroxidase on deoxyribose damage was proportional to the amount of enzyme added (Fig. 4). This eect was observed only when the substrate (cytochrome c) was included in the reaction mixture; a strong indication that the inhibition of TBA-RS formation is due to the enzymatic removal of H2O2 from the reaction mixture and not to heme present in the enzyme added. Figure 5 shows the eect of increasing amounts of FeCl3 on TBARS formation. The protection given by F. hepatica CcP occurred at 10 mM of FeCl3, but not when FeCl3 concentration was increased to 20 and 30 mM. 4. Discussion To investigate the possible causes of the low recovery of enzyme activity during puri®cation, ascorbate was used to reduce the enzyme. For the bacterial di-heme cytochrome c peroxidases, activity seems to be modulated by the oxidation states of the hemes. For example, cytochrome c
peroxidase from P. aeruginosa and P. denitri®cans require reduction of the high potential heme before catalysis can occur [22, 23]. On the other hand, CcP from Nitrosomonas europaea is active in the fully oxidised and partially reduced states [24]. Reduction of the F. hepatica CcP with ascorbate increased the enzyme activity indicating that the oxidative state of the enzyme is important for activity. The increase in F. hepatica CcP activity upon ascorbate reduction was measured in the absence of an electron-transfer mediator in the incubation medium and represents the minimum eect of ascorbate. The eect of calcium on F. hepatica CcP activity was also investigated. Calcium ions are important for horseradish peroxidase activity [25] and seems to be essential for the activation of Paracoccus cytochrome c peroxidase [23]. Yeast cytochrome c peroxidase, in contrast, contains no
Fig. 5. Addition of increasing amounts of FeCl3 to the FeIII± EDTA-ascorbate system increased the formation of TBA-RS and abolished the protection aorded by CcP. Cytochrome c (21 mM) and Cc (21 mM) plus CcP (60 ml) were used. All other reagents in the reaction mixture were the same as described in Fig. 3. The incubation time was 2 h. (A) TBARS formation measured by the absorbance peak at 535 nm. The reduction in TBA-RS formation in the presence of CcP + Cc at a concentration of 10 mM FeCl3 is signi®catively dierent from Cc alone (P = 0.008). (B) Percentage inhibition in relation to the control reaction. Empty bars (CcP + Cc), ®lled bars (Cc alone). Number of determinations, n = 3.
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calcium [26]. In this study calcium seemed not to be involved in F. hepatica CcP activation. In a study of the kinetics of the oxidative deoxyribose degradation reaction catalysed by FeIII±EDTA plus ascorbate, an increase in the amount of FeCl3 added to the reaction (up to 15 mM) promoted a saturable rise in the yields of deoxyribose degradation in the presence of EDTA and ascorbate [16]. This saturation was obtained using 3 mM of ascorbate and 15 mM of deoxyribose compared with 2 and 10 mM, respectively, used in the present study. Based on the results presented in Fig. 5 it can be postulated that the antioxidant activity of CcP is overwhelmed by high levels of H2O2 formation, and consequently hydroxyl radicals, reached at 20 and 30 mM FeCl3. Peroxidases detoxify H2O2, which can damage multiple cellular components. Hydrogen peroxide eects on biological systems have been reviewed by Cochrane [27]. The glycolytic pathway as well as lactate production, for example, were inhibited when P388D1 cells where exposed to H2O2 in vitro [27]. DNA damage has been shown in several cell types which had been exposed to H2O2. The ability of H2O2 to induce DNA strand breaks has been shown to require the presence of FeII, indicating that the mechanism of damage involved the generation of OH from H2O2, through the Fenton Reaction [28]. Thus, H2O2 can penetrate cells and diuse through the cellular compartments, including the nuclei, to cause DNA damage. Therefore, the presence of peroxidases make the cells resistant to DNA damage caused by H2O2. A speci®c physiological function for CcP in F. hepatica has still to be determined, but is most probably linked to the nature and functioning of the intracellular sources of H2O2 and its H2O2 scavenging activity. It has been demonstrated that H2O2 is produced by the mitochondria of adult F. hepatica [8]. Hydrogen peroxide has been considered a normal metabolite in cells, present at steady-state concentrations in the range of 10 ÿ 8 to 10 ÿ 9 M [29]. It can also be formed from superoxide anions which are produced by phagocytic cells [30] and may be present at much higher concentrations at sites of in¯ammation.
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Few studies have dealt with the production of H2O2 by the host immune cells during infections with F. hepatica and the role it may play in resistance or susceptibility to fascioliasis. Smith et al. [31] have found higher production of free radicals by peritoneal leukocytes from infected nonpermissive hosts (rats) compared with leukocytes from infected permissive hosts (mice). On the other hand, BaeÂza et al. [32] found that ES products of F. hepatica caused a decrease in the production of free radicals by bovine polymorphonuclear leukocytes. Thus, it has still to be clari®ed if F. hepatica can inhibit the respiratory burst of host immune cells or if it can detoxify ROS which are formed, avoiding oxidative damage. Any role for cytochrome c peroxidase in protecting F. hepatica or other trematodes, such as S. mansoni [3, 33] from host oxidative attack remains to be determined. Cytochrome c peroxidase might play a role in protection against host immune oxidative attack.
Acknowledgements We acknowledge the contribution of the CIDA/McGill Fellowship Program and the Brazilian Research Council (CNPq/PRONEX), which supported E.G.C. and M.H.L. We are grateful to Christiane Trudeau for her technical assistance and animal care. This project was supported, in part, by a MRC grant to R.K.P. and J.M.S. Research at the Institute of Parasitology is supported by Fonds FCAR pour la Formation des Chercheurs et l'Aide aÁ la Recherche.
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