experimental parasitology - tanta...

Experimental Parasitology 120 (2008) 147–155

Contents lists available at ScienceDirect

Experimental Parasitology

journal homepage: www.elsevier .com/ locate/yexpr

Experimental schistosomal hepatitis: Protective effect of coenzyme-Q10 againstthe state of oxidative stress

Ahmad A. Othman a,*, Zeinab S. Shoheib a, Ghada A. Abdel-Aleem b, Mohamed M. Shareef c

a Department of Parasitology, Faculty of Medicine, Tanta University, Tanta, Egyptb Department of Biochemistry, Faculty of Medicine, Tanta University, Tanta, Egyptc Department of Pathology, Faculty of Medicine, Tanta University, Tanta, Egypt

a r t i c l e i n f o a b s t r a c t

Article history:Received 2 April 2008Received in revised form 9 June 2008Accepted 20 June 2008Available online 1 July 2008

Keywords:Oxidative stressSchistosomaAntioxidantLiver fibrosisCoenzyme-Q10HepatitisHepatic stellate cells

0014-4894/$ - see front matter � 2008 Elsevier Inc. Adoi:10.1016/j.exppara.2008.06.009

* Corresponding author. Fax: +20 2 40 3407734.E-mail address: [email protected] (A.A. O

Schistosoma mansoni (S. mansoni) eggs trapped in the host liver elicit a chain of oxidative processes thatmay be, at least in part, responsible for the pathology and progression of fibrosis associated with schis-tosomal hepatitis. This study was designed to assess the protective effect of the antioxidant coenzyme-Q10 (Co-Q10) against experimental S. mansoni-induced oxidative stress in the liver, and its potential roleas an adjuvant to praziquantel (PZQ) therapy. The oxidative stress and overall liver function wereimproved under Co-Q10 therapy as evidenced by significant reduction in oxidative stress markers andpreservation of antioxidant factors. Liver fibrosis was also reduced with a positive impact on liver func-tion. Moreover, addition of Co-Q10 to PZQ therapy caused: significant reduction of liver egg load, signif-icant improvement of the redox status, and lastly decreased liver fibrosis.

� 2008 Elsevier Inc. All rights reserved.

1. Introduction

Schistosomiasis is one of the leading causes of morbidity andmortality associated with parasitic infections in endemic areas,affecting between 200 and 300 million people in 77 countries allover the world (Curtis and Minchella, 2004). Adult worms of Schis-tosoma mansoni (S. mansoni) reside in the mesenteric venousplexus of the bowel, from where the eggs are frequently sweptback to the liver via the portal blood to induce delayed hypersen-sitivity response (Wynn et al., 2004). The major pathology is gran-ulomatous inflammation which is a cellular response to antigenssecreted by the internal membrane and miracidia of schistosomeeggs. Subsequently, the granulomas heal, resulting in a uniqueform of periportal fibrosis followed by portal hypertension as themain complication (Caulfield et al., 1985; Stavitsky, 2004).Although praziquantel (PZQ) is still considered the treatment ofchoice for human schistosomiasis, there is considerable concernabout its decreased efficacy which may be attributed partly tothe development of drug resistance (Sangster, 2001).

Oxidative stress is a general term used to describe the disturbedbalance between the steady state of reactive oxygen species (ROS)formation in tissues and their efficient consumption by antioxi-dants. Recently, it was proved that the state of oxidative stress at

ll rights reserved.

thman).

the sites of inflammation is responsible to a large extent for cellulardamage and progression of fibrosis in chronic liver diseases includ-ing schistosomal hepatitis (Ames et al., 1993; Gharib et al., 1999).Activated neutrophils, eosinophils, macrophages, and Kuppfer cellsare major sources of ROS in the vicinity of schistosome eggs duringinflammation. Eosinophils, which are abundant in Schistosoma-in-duced hepatic granulomas, generate reactive oxygen (O2

�) and hy-droxyl (OH�) radicals following stimulation. Also, hydrogenperoxide (H2O2) is released in significant amounts by the macro-phages. As a result, the endogenous antioxidants such as glutathi-one and glutathione peroxidase are depleted, and oxidative stressensues (Abdallahi et al., 1999; Kaplowitz, 2000).

Hepatic stellate cells (HSCs) play a major role in the process offibrous tissue formation in the liver. It was found that these cellsare more vulnerable to the oxidative stress-related molecules ow-ing to their low levels of endogenous antioxidant enzymes (Parolaand Robino, 2001). This oxidative stress presents a direct or indi-rect relevant profibrogenic stimulus for HSCs where signs of oxida-tive stress and lipid peroxidation were found to be concomitant orprecede HSC activation and collagen deposition (Tsukamoto et al.,1995). Therefore, antioxidant treatment in vivo was found to beeffective in preventing or reducing liver fibrosis, as demonstratedin several experimental models (Pietrangelo et al., 1995; Lieber,2000; Loguercio and Federico, 2003).

The normal liver is a well equipped organ in terms of antioxi-dant mechanisms either enzymatic such as catalase, glutathione

mailto:[email protected]

http://www.sciencedirect.com/science/journal/00144894

http://www.elsevier.com/locate/yexpr

148 A.A. Othman et al. / Experimental Parasitology 120 (2008) 147–155

peroxidase, and glutathione, or non-enzymatic ones such asa-tocopherol, ubiquinol, and ascorbic acid (Parola and Robino,2001; Zhen et al., 2007). Moreover, paraoxonase and arylesterasewhich are detoxifying hepatic enzymes were proved to have anti-oxidant activities. Recently, both enzymes were proved useful asbiomarkers of liver function (Gangadharan et al., 2007; Campset al., 2007). Nitric oxide (NO�) is a short-lived gaseous free radicalwhich can act either as a cytoprotective or a cytotoxic agent. Pro-tective effects, for example reducing the proliferative responsesof activated HSCs, predominate in the presence of low levels ofROS (Grisham et al., 1998; Carnovale et al., 2000).

Coenzyme-Q10 (Co-Q10), also known as ubiquinone, is a vita-min-like substance present in all human cells in the membranesof endoplasmic reticulum, peroxisomes, lysosomes, and the innermembrane of mitochondria. It is an important component of theelectron transport chain in mitochondria responsible for genera-tion of energy. Furthermore, its reduced form serves as an impor-tant antioxidant in both mitochondria and lipid membraneswhere it protects the body against the deleterious effects of ROS.Therefore, the enzyme has been therapeutically employed in manydisorders for its cytoprotective and antioxidant properties (Allevaet al., 2001; Ruiz-Jimenez et al., 2007).

The aim of the present study was to investigate the effect of Co-Q10 on the state of oxidative stress during Schistosoma-inducedhepatitis in an experimental animal model. Further, the potentialrole of this coenzyme as an adjuvant to the classic anthelminthictreatment was to be assessed.

2. Materials and methods

2.1. Parasite

Laboratory bred Biomphalaria alexandrina snails were purchasedfrom the Schistosome Biological Supply Program, Theodore BilharzResearch Institute (Giza, Egypt). After exposure to light for at least4 h, S. mansoni cercariae shed from the snails were used to infectthe experimental animals of the study.

2.2. Drugs

Co-Q10 (MEPACO, Egypt) was presented as 30 mg tablets. Dailyoral dose for each animal was 5 mg/kg-body weight according toSingh et al. (2000). PZQ (Bayer) was available as Biltricide600 mg tablets. It was given orally in a dose of 300 mg/kg-bodyweight according to Ismail et al. (1996).

2.3. Experimental design

One hundred and sixty, laboratory bred and parasite free male Swissalbino mice ‘‘6 weeks old and 20–25 g weight” were included in thisstudy. Ten mice were left uninfected and served as the non-infectednon-treated control for biochemical parameters and immunohisto-chemical staining (group N). The remaining animals were infected sub-cutaneously with S. mansoni cercariae ‘‘60 ± 10 cercariae/animal”(Peters and Warren, 1969), then divided into four main groups:

Group I included 50 mice that served as infected non-treatedcontrol group for comparison in different durations of infection:8 and 12 weeks post-infection (p.i.).

Group II included 50 mice that received Co-Q10 daily from thefirst day of infection, and sacrificed at 8 and 12 weeks p.i.

Group III included 25 mice that received single oral dose of PZQat the 8th week p.i. and sacrificed 4 weeks later.

Group IV included 25 mice that received PZQ therapy at the 8thweek p.i., and also received Co-Q10 at the same time for four suc-cessive weeks (till the 12th week p.i.).

Mice were purchased from Theodore Bilharz Research Institute(Giza, Egypt) and were housed and infected in accordance with theinstitutional guidelines. At 8 weeks p.i., half animals of groups (I),(II) and (N) were sacrificed and all the remaining animals were sac-rificed at 12 weeks p.i. At both times, the liver of each mouse wasimmediately removed and divided into three portions for parasito-logical, biochemical and histopathological studies.

2.4. Parasitological study

Liver egg load was estimated in all groups. One gram from eachliver was weighed, and then put in test tube containing 2 ml of 5%KOH and left overnight at room temperature. The second day, alltest tubes were put in the incubator at 37 �C for 6 h. Each test tubewas shaken then 0.1 ml of the digest was examined microscopi-cally for counting S. mansoni eggs. The total egg count in 1 g livertissue was then calculated (Cheever, 1968).

2.5. Biochemical study

This was carried out on both tissue and serum samples. Mal-ondialdehyde, and nitric oxide were measured as oxidative stressmarkers, while glutathione, paraoxonase-1, and arylesterase weremeasured as endogenous antioxidant factors. All chemicals usedwere of high analytical grade, products of Sigma (USA), Merckand Reidel (Germany), BDH (England), and Fluka (Switzerland)Chemical Co.

2.5.1. Tissue samplesA part of the liver of each mouse was removed, washed with sal-

ine, blotted with filter paper and weighed. Liver samples werehomogenized in phosphate buffer saline 10 mM, pH 7.4 (20% w/v), using potter-Elvehjem tissue homogenizer. The crude homoge-nate was centrifuged at 7700 g for 30 min at 4 �C, and the resultantsupernatant, free of insoluble materials, was essayed for proteincontent (Bradford, 1976). Aliquots were adjusted to contain1 mg/ml protein to make the protein content of the samples fixedthroughout the experiment and aliquots were assayed for the fol-lowing parameters.

2.5.1.1. Estimation of glutathione. Glutathione was estimated by themethod of Moron et al. (1979) using sodium phosphate buffer and2 mM dithiobisnitrobenzoic acid (DTNB). The developed color wasread against blank at 412 nm within 5 min in spectrophotometer.The amount of glutathione was calculated as lmol/mg tissue pro-tein used from a standard curve plotted for serial concentrations ofglutathione (5–100 lg).

2.5.1.2. Estimation of paraoxonase-1 activity. Paraoxonase activitywas determined by using paraoxon (O,O-diethyl-O-p-nitrophenylphosphate) (1 mM) as a substrate. The initial rate of substratehydrolysis to p-nitrophenol during 5 min was recorded as parao-xonase activity using spectrophotometer at 405 nm. The enzymeactivity was expressed in U/mg; where 1 unit of enzyme hydro-lyzes 1 nmol of paraoxon/min/ml (Ferré et al., 2002).

2.5.1.3. Estimation of arylesterase activity. Arylesterase activity wasmeasured using 5 nmol/l of phenylacetate as a substrate. The rateof formation of phenol during 5 min was recorded as arylesteraseactivity using spectrophotometer at 270 nm. The enzyme activitywas expressed in U/mg; where 1 unit of enzyme hydrolyzes 1 nmolof phenylacetate/min/ml (Lorentz et al., 1979).

2.5.1.4. Estimation of lipid peroxides. Lipid peroxides were esti-mated by thiobarbituric acid reaction using saturated thiobarbitu-ric acid and trichloroacetic acid (20%) solutions. The developed

0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000

I 8wk I 12wk II 8wk II 12wk III IV

Fig. 1. Mean liver egg counts. Group I: infected control, group II: treated by Co-Q10,group III: treated by PZQ, and group IV: treated by combined therapy (Co-Q10 + PZQ).

A.A. Othman et al. / Experimental Parasitology 120 (2008) 147–155 149

color was read against a blank at 530 nm in spectrophotometer andcalculated as nmole malondialdehyde (MDA)/mg tissue (Ohkawaet al., 1979).

2.5.1.5. Estimation of nitric oxide (NO�). Griess method has been con-ducted for micro-determination of nitrite and nitrate (NO2

�, NO3�).

This procedure, supplemented with deproteinization and reduc-tion of nitrate to nitrite in the presence of NADPH-sensitive nitratereductase (EC.1.6.6.2), was followed by spectrophotometric assayat wave length 540 nm (Ding et al., 1988).

2.5.2. Serum samplesUsed to measure levels of liver enzymes: alanine aminotrans-

ferase (ALT) and aspartate aminotransferase (AST) as liver functiontests (Wilkinson et al., 1972).

2.6. Histopathological and immunohistochemical study

Samples of liver tissue were collected and fixed overnight inneutral buffered formalin [10%] and processed for paraffin embed-ding. Two paraffin sections (each of 4 lm thick) per slide, sepa-rated by 20 intervening sections, each 10 lm thick, were cut andthe following stains were used:

� Haematoxylin and eosin (H&E) stain for routine examination.Digital photos of representative areas were captured by Olym-pus digital camera (E 330) installed on Olympus microscopeCX31. The maximal diameters of the granulomas were measuredby image analysis software [Micrometrics SE/CMOS. Version2.6]. The granulomas were chosen so that each measured gran-uloma was discrete (non-confluent) and showing a central Schis-tosoma egg. Ten granulomas in each section were measured, andthen the mean diameter of each group was calculated.

� Masson trichrome stain for demonstration of type of granuloma.The composition of the granulomas was assessed and the gran-ulomas were divided subjectively into three types (fibrous, cel-lular, and fibrocellular) according to the predominant cellularcomponent (Costa-Silva et al., 2002).

� Immunohistochemical identification of activated hepatic stellatecells (HSCs) by immuno-staining for a-smooth muscle actin [a-SMA]. The immuno-stain was done on randomly selected paraffinblocks from each group [2 blocks from group N and 5 blocks fromeach infected group]. The immuno-stain was done as follows: sec-tions were deparaffinized in xylene then rehydrated in descend-ing grades of alcohol. The endogenous peroxidase positivity wasblocked by incubation in 5% hydrogen peroxide in methanol.Microwave antigen retrieval was carried out in citrate buffer. Sec-tions were flooded by the primary antibody [Mouse MonoclonalAntibody against a-SMA Ab-1, NeoMarkers, Fermont, California(Cat. #MS-113-P)] for 2 h and then washed by phosphate buffer.Sections were then treated by the secondary antibodies followedby the avidin–biotin complex and the DAB chromogen. Sectionswere then dehydrated and mounted in synthetic resin. Negativecontrol was obtained by omitting the primary antibodies, andinternal positive control was the a-SMA positive vascular smoothmuscle fibers in the vessels of the portal tracts. The immuno-staining assessment was carried out in a blinded subjectivemethod for the overall positivity for each section: (+++) for intensepositivity, (++) for moderate positivity, (+) for mild positivity and(�) for negative staining.

2.7. Statistical analysis

Data were presented as means ± standard deviation. The proba-bility of significant differences among dual means of groups was

determined by Student’s t-test. Differences were considered non-significant when (P > 0.05), significant (P < 0.05), and highly signif-icant (P < 0.001). The statistical analyses were processed accordingto the conventional procedures using Statistical Program of SocialSciences (SPSS) software for windows, version10.0.

3. Results

3.1. Hepatic egg load

Results of liver egg load were shown in Fig. 1. There were no sig-nificant differences in the mean numbers of liver egg count in thegroup that received Co-Q10 and the control one. Egg counts 8weeks p.i. were (4301.6 ± 512.5 vs 3842.8 ± 532; P = 0.061) andwere (4861.3 ± 786.9 vs 4350.1 ± 761.2; P = 0.157) at 12 weeksp.i. Both group (III) that received PZQ therapy and group (IV) thatreceived combined therapy showed highly significant decrease(P = 0.000) in mean numbers of eggs trapped in the liver comparedto the control group: (1245.9 ± 345.2 and 512.1 ± 191.3 vs4350.1 ± 761.2), respectively. Also, statistically significant reduc-tion of liver egg count (P = 0.003) was found between groups (IV)and (III).

3.2. Biochemical parameters

Results of biochemical study 8 weeks p.i. were demonstrated inTable 1. Glutathione level and activity of paraoxonase and arylest-erase were significantly reduced whereas levels of MDA and NO�

were significantly increased (P < 0.05) in infected control animalsin comparison to the non-infected non-treated mice (group N).Administration of Co-Q10 for 8 weeks had a statistically significantpositive impact. Measured antioxidant factors were increased andlevels of oxidative stress markers were reduced; however, differ-ences with group (N) were statistically significant (P < 0.05). Also,comparison between group (II) and group (I) as regards the previ-ously mentioned values showed significant differences (P < 0.05).Moreover, serum liver enzymes (ALT, AST) were significantly in-creased (P < 0.05) in group (I) in comparison to group (N). This in-crease was reduced significantly in group (II).

Table 2 shows results of biochemical parameters 12 weeks p.i.Significant reduction (P < 0.05) of values of antioxidant parameterswith significant increase of oxidative stress markers in group (I) incomparison to group (N) was recorded. Administration of Co-Q10for 12 weeks had a normalizing effect on both antioxidant factorsand oxidative stress markers, where their values in group (II)became non-significantly different (P > 0.05) from those of the

Table 1Values (mean ± SD) of biochemical parameters 8 weeks post-infection

Group Liver homogenate Serum

Glutathione(lmol/mg)

Paraoxonase(U/mg)

Arylesterase(U/mg)

Malondialdehyde(nmol/mg)

Nitric oxide(nmol/mg)

ALT(U/ml)

AST(U/ml)

N 1.71 ± 0.04 22 ± 0.75 264 ± 8.9 30 ± 0.95 27.1 ± 2.5 22 ± 2.92 20 ± 2.11Non-infected non-treated controlI 1.079 ± 0.15 18 ± 0.85 255 ± 8.25 41 ± 3.68 39.76 ± 2.31 31 ± 5.3 23 ± 3.8Infected non-treatedII 1.503 ± 0.13 19 ± 1.51 260 ± 4.50 35.3 ± 1.05 31.3 ± 1.55 25 ± 2.45 19 ± 0.85Infected received Co-Q10P1 (I vs N) 0.000 0.000 0.050 0.002 0.000 0.000 0.042P2 (II vs N) 0.025 0.003 0.046 0.002 0.001 0.519 0.056P3 (I vs II) 0.005 0.001 0.005 0.001 0.001 0.004 0.028

P > 0.05 ? non-significant, P < 0.05 ? significant, P < 0.001 ? highly significant.

Table 2Values (mean ± SD) of biochemical parameters 12 weeks post-infection

Group Liver homogenate Serum

Glutathione(lmol/mg)

Paraoxonase(U/mg)

Arylesterase(U/mg)

Malondialdehyde(nmol/mg)

Nitric oxide(nmol/mg)

ALT(U/ml)

AST(U/ml)

N 1.71 ± 0.04 22 ± 0.75 264 ± 8.9 30 ± 0.95 27.1 ± 2.5 22 ± 2.92 20 ± 2.11Non-infected non-treated

controlI 0.98 ± 0.13 16.33 ± 1.52 243 ± 8.3 49 ± 3.74 42.36 ± 5.241 37 ± 3.7 26 ± 6.2Infected non-treatedII 1.78 ± 0.05 21 ± 0.49 267 ± 5.8 32 ± 1.11 26.1 ± 0.95 24 ± 2.95 18 ± 0.84Infected received Co-Q10III 1.62 ± 0.04 20 ± 1 251.6 ± 8.08 35 ± 1.3 33 ± 1.1 29 ± 1.8 19 ± 0.92Infected received PZQIV 1.74 ± 0.06 23.3 ± 1.52 277 ± 10.53 30.3 ± 2.08 25.7 ± 0.85 20 ± 2.2 15 ± 2.1Infected received Co-Q10

and PZQP1 (II vs N) 0.515 0.148 0.243 0.136 0.348 0.628 0.058P2 (IV vs N) 0.110 0.126 0.212 0.529 0.348 0.057 0.004P3 (IV vs III) 0.001 0.017 0.047 0.004 0.001 0.001 0.002

P > 0.05 ? non-significant, P < 0.05 ? significant, P < 0.001 ? highly significant. Differences between group (I) and (N and IV) were statistically highly significant, and weresignificant between group (I) and (II and III). Also, differences between groups (III) and (N) were statistically significant.


normal ones (group N). The same changes were found as regardsserum liver enzymes (ALT, AST). Comparison between group (I)and group (II) as regards all the measured biochemical parametersrevealed significant differences (P < 0.05). Praziquantel therapy(group III) had a positive impact on both antioxidant and oxidativestress values when compared to infected control (group I) with sta-tistically significant differences (P < 0.05). Addition of Co-Q10 tothe classic therapy (group IV) showed much better improvementin the measured values, where differences with the normal onesbecame statistically non-significant (P > 0.05). Levels of serum liverenzymes were improved significantly in group (IV) in comparisonto group (III).

3.3. Histopathological findings

Histopathological examination studied both size (expressed asmean diameter) and type (cellular, fibrocellular or fibrous) of thegranulomas. Mean diameter of granulomas of all groups were dem-onstrated in Table 3. There was a significant reduction (P < 0.05) in

Table 3Values (mean ± SD) of diameter of schistosomal granuloma

Group I I IIInfected non-treated8 wk p.i.

Infected non-treated12 wk p.i.

InCo

Diameter of granulomasin lm

465.44 ± 9.4 408.8 ± 8.5 35

P value II

P > 0.05 ? non-significant, P < 0.05 ? significant, P < 0.001 ? highly significant.

the mean diameter of granulomas of group (II) (Fig. 5) in compar-ison to group (I) (Figs. 3 and 4) at both durations of infection. Also,there was a highly significant reduction (P < 0.001) in the meandiameter of granulomas of group (III) in comparison to group (I).The combined therapy caused more reduction in mean diameterof granulomas with statistically significant difference (P < 0.05) be-tween groups (IV) and (III).

Differential types of granulomas of the studied groups wereillustrated in Fig. 2. At 8 weeks p.i., most of the granulomas ingroup (II) were of the cellular type 67% with only 10% fibrous ones.The reverse was observed in group (I) where fibrous granulomasrepresented 53% vs 8% of the cellular type. At the second timep.i. group (II) showed also less fibrous granulomas 30% vs 83% ingroup (I) and higher percentage of cellular granulomas 49% vs 5%in group (I). As regards to group (IV) there was decreased percent-age of fibrous granulomas 45% in comparison to 67% in group (III).Also, the cellular granulomas in group (IV) were increased in com-parison to group (III), where the calculated percentages were 29%and 12%, respectively.

II III IVfected received-Q10 for 8 wk

Infected receivedCo-Q10 for 12 wk

Infectedreceived PZQ

Infected receivedPZQ and Co-Q10

9.32 ± 12.2 326 ± 23.5 288.9 ± 14.9 222 ± 24.9

vs I 0.001 II vs I 0.002 III vs I 0.000 IV vs I 0.000 IVvs III 0.005

0%

20%

40%

60%

80%

100%

I 8wk II 8wk I 12wk II 12wk III IV

Fibrous Fibrocellular Cellular

Fig. 2. Percentage of different types of granulomas. Highest percentage of fibrousgranulomas was recorded in group (I) 12 weeks p.i. and the lowest percentage wasin group (II) 8 weeks p.i. For cellular granulomas, the highest percentage is in group(II) and the lowest in group (I).

Fig. 3. Liver section of control group 12 weeks p.i. Many bilharzial granulomas oflarge size and fibrous type are seen (arrow heads) [H&E 160�, bar: 200 lm].

Fig. 4. Liver section of control group 12 weeks p.i., showing mature fibrousgranuloma [Masson Trichrome 400�].

Fig. 5. Liver section of Co-Q10 treated group 12 weeks p.i. Many small-sizedcellular bilharzial granulomas (arrow heads) and few fibrous granulomas (arrows)can be observed [H&E 160�, bar: 200 lm].

Fig. 6. Liver section of control infected group 12 weeks p.i. There is intensecontinuous sinusoidal wall positivity (+++) for a-SMA [400�, immunoperoxidase a-SMA].

Fig. 7. Liver section of PZQ treated group. Focal intense sinusoidal wall positivity isseen adjacent to the granuloma with some positive cells in the granuloma (asterisk)[400�, immunoperoxidase a-SMA].


3.4. Immunohistochemical localization of a-SMA

Results of immunohistochemical staining of hepatic stellatecells (HSCs) agreed with those of granuloma type study. The stain-ing in the normal control group (group N) was limited to the vas-cular walls in the portal areas and to the large and medium sizedhepatic venules. The highest positivity of the stain (+++) was ob-

served in the non-treated infected control group, where intensea-SMA positivity was detected diffusely in the walls of sinusoids,in portal tracts, and areas of fibrosis (Fig. 6). In group (III) the walls

Fig. 8. Liver section of Co-Q10 treated group 12 weeks p.i. Mild discontinuoussinusoidal wall positivity (+) for a-SMA is seen [400�, immunoperoxidase a-SMA].

Fig. 9. Mild positivity adjacent to the granuloma in Co-Q10 treated group [lessfibrous, smaller and more cellular than in PZQ group], and some positive cells insidethe granuloma (asterisk) [400�, immunoperoxidase a-SMA].

Fig. 10. Very faint sinusoidal wall staining is noticed adjacent to the granuloma(asterisk) which showed some stained cells in combined therapy group [400�,immunoperoxidase a-SMA].

Fig. 11. Liver section of combined therapy group showing almost completenegative sinusoidal staining of lobular areas [400�, immunoperoxidase a-SMA].


of the sinusoids showed continuous moderate positivity (++) withmore prominent staining adjacent to the granulomas (Fig. 7). Co-Q10 strikingly decreased stellate cell activation, where group (II)showed only mild staining intensity (+) with discontinuous mildsinusoidal wall positivity (Figs. 8 and 9). The least staining inten-

sity (+ or �) was detected in group IV (Fig. 11). Very faint positivitywas detected in the wall of the sinusoids close to the granulomastogether with few cells in the granulomas themselves (Fig. 10).

4. Discussion

Schistosomiasis is an important public health disease in manydeveloping countries including Egypt. Hepatosplenic schistosomia-sis is a serious manifestation of S. mansoni infection that may leadto irreversible sequelae (Sayed et al., 2004). Recent research stres-ses the role of free radicals and oxidative stress in progression ofliver injury in various chronic liver diseases such as viral hepatitis,alcoholic hepatitis, and cirrhosis (Parola and Robino, 2001; Loguer-cio and Federico, 2003). Schistosomiasis is no exception: oxidativestress occurs in the liver at the site of inflammation in the vicinityof eggs of S. mansoni. This state of oxidative stress is attributed toincreased generation of ROS and exhaustion of endogenous antiox-idant enzymes (Gharib et al., 1999). Therefore, it is tempting to testthe efficacy of exogenous antioxidants as an adjuvant therapy inchronic liver diseases including schistosomiasis.

In the present study we aimed at investigating the protectiverole of Co-Q10 against the oxidative stress induced by experimen-tal S. mansoni infection, and its potential role in therapy along withthe classical treatment. Co-Q10 is an endogenous antioxidant andan important component of mitochondrial metabolism (Ruiz-Jime-nez et al., 2007). It is available commercially in many countries asan over the counter dietary supplement, and is used therapeuti-cally in heart failure, cardiomyopathy, neurodegenerative disor-ders as well as in diabetes. All these disorders have in commonthe presence of oxidative stress that damages the tissues. It is note-worthy that the drug is safe, even in high doses, with few reportedside effects (Crane, 2001; Miles et al., 2007).

The effect of Co-Q10 on schistosomal hepatitis 8 and 12 weeksp.i. was investigated. As regards the egg count in the liver, no sig-nificant effect at both times was recorded compared to control in-fected animals. Meanwhile we found significant reduction inoxidative stress markers: MDA and NO�, and significant increasein endogenous antioxidant factors, namely level of glutathione,and activity of paraoxonase and arylestrase in animals that re-ceived Co-Q10 compared with infected control groups at bothtimes p.i.

This study demonstrated that Co-Q10 improved the redoxmetabolism of the liver as manifested by reduction of oxidativestress markers and preservation of the endogenous antioxidantmechanisms. This could be explained by inactivation of ROS, gen-erated during the process of inflammation, by the antioxidant


activity of the drug. The significant decrease of serum level of ASTand ALT further confirms the beneficial effect of controlling theoxidative stress on the overall liver function. As Co-Q10 has severalcytoprotective functions, the possibility exists that the drug exertsadditional protective effect by other mechanisms which are yet inneed for further investigation.

Interestingly, at 8 weeks p.i. the mean diameter of granulomaswas reduced in the treated group with Co-Q10. The granulomas inthe control group were mostly fibrocellular, while in Co-Q10-receiving mice they were mostly cellular. At 12 weeks p.i thesechanges were less prominent, probably due to the process ofimmunomodulation that occurs normally in chronic schistosomia-sis where the eggs deposited late induce smaller granulomas thanthose deposited earlier in the course of infection (Wilson et al.,2007). The situation seems to be beneficial as increased cellularityof early granulomas is presumably detrimental to the eggs, whilethe host is protected by the reduction of redox metabolism ensuredby Co-Q10.

Many studies implemented various antioxidant strategies toprotect against oxidative stress in schistosomiasis. One studyemployed melatonin and found significant reduction of hepaticredox metabolism. Melatonin caused reduction of lipid oxida-tion products and NO�, and increase in vitamin E, catalase,and superoxide dismutase in the liver. The authors attributedthese effects to the antioxidant activity of melatonin (El-Sokkaryet al., 2001). Similarly, Gharib et al. (2001) have demonstratedthat S. mansoni-induced liver inflammation and fibrosis isclearly associated with generation of ROS and depletion of anti-oxidant enzymes. They further employed molecular hydrogen—apotent antioxidant—which exerted significant protective effectson the liver, namely reduction in fibrosis, lipid peroxide levels,and tumor necrosis factor-a; and increase in antioxidant en-zyme activity.

Antioxidants, both natural and synthetic, have been also used astherapeutic agents in liver damage. In vitro and in vivo activation ofKupffer cells, collagen gene expression, hepatitis B virus (HBV) andhepatitis C virus (HCV) replication, were countered by N-acetyl-cysteine, selenium, vitamin C and E (Lieber, 2000; Soltys et al.,2001; Turkodgan et al., 2001).

Fibrosis is the ultimate sequel of chronic schistosomal hepatitis.A characteristic type of severe periportal fibrosis occurs, whichleads to presinusoidal portal hypertension and esophageal varices(Stavitsky, 2004). One of the major goals in therapy of S. mansoniis to block the pathways that lead ultimately to liver fibrosis. Thehepatic stellate cells (HSCs) play a major role in this process as theyare responsible for synthesis of components of extra-cellular ma-trix and several types of collagen (Parola and Robino, 2001). Thedifferent stimuli that initiate and perpetuate HSC activation inchronic liver diseases are poorly understood, but recent studiesstress the role of ROS as activators of HSCs and question the poten-tial of various antioxidants at halting the progression of fibrosis(Loguercio and Federico, 2003).

In this study, fibrosis was assessed by Masson trichrome stain-ing, and the activity of HSCs was assessed by a-smooth muscle ac-tin immunohistochemical staining. Evident reduction of activitywas observed in groups that received Co-Q10 as compared to con-trol infected groups notably at 12 weeks p.i. We assume that Co-Q10 reduced the oxidative stress at the site of inflammation, thusshut down the stimulatory pathways of HSC activation. Shoheib(2002) has demonstrated reduction in the thickness of the capsulesaround Trichinella spiralis (T. spiralis) encysted larvae in rats underCo-Q10 therapy, denoting decreased collagen deposition. Further,addition of Co-Q10 to mebendazole during the muscular phase ofinfection enhanced the efficacy of the latter. Larval counts were re-duced and the capsules were damaged compared to animalsreceiving mebendazole only.

The antifibrotic effect of antioxidants has been explored in sev-eral studies whether employing a-tocopherol (vitamin E), or theflavinoids sylimarin (Bedossa et al., 1994; Boigk et al., 1997). More-over, the antifibrogenic activity displayed by estrogens on di-methyl-nitrosamine-induced hepatic fibrosis is mediated, at leastpartly, by an antioxidant mechanism (Yasuda et al., 1999). Trialsin humans are relatively few, and they have been performed usingvery different doses or therapeutic combinations for quite limitedperiod of time and on patients with long standing chronic liver dis-ease. However, encouraging results have been obtained with a-tocopherol (Ferro et al., 1999).

Nitric oxide (NO�) is an ambivalent agent that acts as a mediatorof inflammation and as a regulator of redox metabolism. In absenceof oxidative stress, it acts as an endogenous antioxidant (Grishamet al., 1998). Remarkably, nitric oxide can inhibit the initiation orpropagation of lipid peroxidation by rapidly reacting with a num-ber of ROS including lipid alkoxyl (LO�) and lipid hydroxperoxyl(LOO�) radicals which are generated as intermediates during lipidperoxidation (Parola and Robino, 2001). In this experiment Co-Q10 caused reduction of oxidative stress, thus favoring the antiox-idant activity of NO�.

Moreover, relatively high levels of NO� in the tissues were ob-served in control infected animals, and this may be responsible,in part, for the recruitment of inflammatory cells in the granulo-mas. Therefore, reduced NO� levels in the tissues under Co-Q10therapy may be responsible for the reduced size of granulomas.Interestingly, recent data seem to indicate that NO� may block orreduce proliferative response of activated HSCs. Nitric oxide canefficiently inhibit prostaglandin F-dependent proliferation andchemotaxis in activated human HSCs. These effects are more pro-nounced in lack of oxidative stress (Failli et al., 2000). So, it seemsthat Co-Q10 provides a favorable background for the beneficial ef-fects of NO� to appear.

Recently, there has been an increasing interest in paraoxonaseand arylesterase as potential biomarkers for chronic liver impair-ment (Camps et al., 2007; Kilic et al., 2005; Aslan et al., 2007). De-creased activities of both enzymes may contribute to liverdysfunction by effect on lipid metabolism and peroxidation (Aslanet al., 2007). Moreover, paraoxonase plays a role in the regulationof oxidative stress and hepatic cell apoptosis—events that are clo-sely related to liver fibrosis. Paraoxonase activity is thus decreasedin patients with chronic hepatitis or liver fibrosis (Camps et al.,2007). In the present study, the infected control group showedreduction in the activity of both enzymes in the liver, which wasreversed significantly by treatment with Co-Q10. These findingsare consistent with those of Ali (2004) who found that the activi-ties of paraoxonase and arylesterase were partially restored in liverand serum by chronic administration of zinc as compared to con-trol mice in experimental S. mansoni model.

We investigated the role of Co-Q10 as a potential adjuvant inschistosomal therapy where PZQ was given once in a dose of300 mg/kg and Co-Q10 in a dose of 5 mg/kg-body weight for onemonth. Significant reduction of hepatic egg count was observedin mice that received combined therapy (PZQ + Co-Q10) in compar-ison to either, animals that received PZQ alone or control non-trea-ted ones. The reason here is not clear, but a possible explanation isreduction in fibrosis of the intestine, allowing more eggs to pass inthe stool instead of being swept away to the liver. We actually ob-served reduction in fibrosis of the colon in histopathological sec-tions under the effect of Co-Q10 (data not shown). However,further confirmation is needed.

As regards the biochemical parameters, significant reduction inoxidative stress markers in combined therapy as compared withcontrol non-treated and PZQ treated groups was found. Free radicalgeneration was reduced, evidenced by reduction in MDA and NO�

levels, while endogenous antioxidant factors, namely glutathione


level and activities of paraoxonase and arylesterase were in-creased. Serum levels of ALT and AST were significantly decreasedunder combined therapy denoting diminished tissue injury andimproved liver functions.

Histopathological examination of liver sections during the cur-rent study revealed significant reduction in the mean size of gran-ulomas under combined therapy compared to control mice andthose that received PZQ only. Furthermore, Co-Q10 combined withPZQ exhibited a potent antifibrotic activity via its inhibitory effecton HSCs as confirmed by immunohistochemical assessment of a-SMA. Apparently, the drug produced rapid healing of granulomasand checked the progression of liver fibrosis. Hepatic stellate cellsplay a key role in initiation and progression of liver fibrosis andwere probably inhibited by the improvement of redox metabolismunder Co-Q10 therapy. Moreover, these cells affect adversely thehepatic microcirculation; on activation, they transform into myofi-broblasts that contract around the sinusoids, increasing the vascu-lar resistance and contributing to the development of portalhypertension (Friedman, 2000). Therefore, by inhibiting HSC activ-ity, a second hepato-protective role of Co-Q10 is confirmed in ourstudy.

Studies of antioxidants in parasitic infections have sometimesyielded equivocal or even discouraging results. Many factors inter-vene such as the habitat of parasites, stage and intensity of infec-tion, immune status, and presence of co-infections. Salem and El-Sheikh (1998) have found increased T. spiralis larval burden undertherapy with exogenous antioxidants. Furthermore, Daoud et al.(2000) have found that administration of exogenous antioxidantpreparation (Antox) in T. spiralis-infected mice lead to delayedexpulsion of adults, increased muscle larval burden, and decreasedefficacy of mebendazole therapy in the intestinal phase. In con-trast, mebendazole therapy was enhanced by the antioxidant dur-ing the muscular phase of infection. The antioxidants seem toexhibit a dual role; on the one hand they reduce the oxidativestress that is detrimental to the host, on the other, they may pro-tect the parasite vis-à-vis the host defense mechanisms. The finalbalance is a quite delicate process.

Fortunately, in the present work, Co-Q10 exerted protective ef-fect against the deleterious inflammation and fibrosis in experi-mental schistosomiasis despite lack of effect on the intensity ofinfection. In contrast, with PZQ the drug exerted protective effecton the liver and decreased the intensity of infection. It seems thatthe drug enhances the anti-parasitic activity of PZQ. These resultsare highly relevant especially with the multitude of reports ofincreasing resistance to PZQ.

In conclusion, this study has demonstrated for the first time, tothe best of our knowledge, a protective effect of Co-Q10 on the li-ver against the state of oxidative stress induced by S. mansoniinfection. Liver fibrosis in the groups that received this drug wasalso greatly reduced. Moreover, Co-Q10 enhanced the efficacy ofanti-schistosomal therapy. Therefore, Co-Q10 could be consideredas a useful adjuvant treatment in hepatic schistosomiasis mansoni,and further studies and controlled human trials on it are worthconsideration in schistosomiasis and perhaps other chronic liverdiseases.

References

Abdallahi, O.M., Hanna, S., de-Reggi, M., Gharib, B., 1999. Visualization of oxygenradical production in mouse liver in response to infection with Schistosomamansoni. Liver 19, 495–500.

Ali, M.E., 2004. Arylesterase and paraoxonase in serum and liver of mice infectedwith Schistosoma mansoni: effect of zinc administration. Egyptian Journal ofBiochemistry 22, 123–138.

Alleva, R., Tomasetti, M., Battino, M., Curatola, G., Littarru, G.P., Folkers, K., 2001. Theroles of coenzyme-Q10 and vitamin E on peroxidation of human low densitysub-fractions. Proceedings of the National Academy of Sciences of United Statesof America 92, 9388–9391.

Ames, B.N., Shinenago, M.K., Hagen, T.M., 1993. Oxidants, antioxidants anddegenerative diseases of aging. Proceedings of the National Academy ofSciences of United States of America 90, 7915–7922.

Aslan, M., Horoz, M., Nazligul, Y., Bolukbas, C., Bolukbas, F.F., Selek, S., Aksoy, N.,Erel, O., 2007. Serum paraoxonase and arylesterase activities for the evaluationof patients with chronic hepatitis. International Journal of Clinical Practice.Epub ahead of print.

Bedossa, P., Houglum, K., Trautwein, C., Holstege, A., Chojkier, M., 1994. Stimulationof collagen-a (I) gene expression is associated with lipid peroxidation inhepatocellular injury. A link to tissue fibrosis? Hepatology 19, 1262–1271.

Boigk, G., Stroedter, L., Herbst, H., Waldschmidt, J., Riecken, E.O., Schuppan, D., 1997.Silymarin retard collagen accumulation in early and advanced biliary fibrosissecondary to complete bile duct obliteration in rats. Hepatology 26, 643–649.

Bradford, M.M., 1976. A rapid and sensitive method for the quantitation ofmicrogram quantities of protein utilizing the principle of protein dye binding.Analytical Biochemistry 72, 248–254.

Camps, J., Marsillach, J., Joven, J., 2007. Measurement of serum paraoxonase-1activity as a potential biomarker for chronic liver impairment. Clinica ChimicaActa 386, 114–115.

Carnovale, C.E., Scapini, C., Alvarez, M., Favre, C., Monti, J., Carillo, M.C., 2000. Nitricoxide release and enhancement of lipid peroxidation in regenerating rat liver.Journal of Hepatology 32, 798–804.

Caulfield, J.P., Lenzi, H.L., Elsas, P., Dessein, A.G., 1985. Ultra-structure of the attackeosinophils stimulated by blood mononuclear cell products on schistosomula ofSchistosoma mansoni. American Journal of Pathology 120, 380–390.

Cheever, A.W., 1968. Conditions affecting the accuracy of potassium hydroxidedigestion in techniques for counting S. mansoni eggs in tissues. Bulletin WHO39, 328–331.

Costa-Silva, M., Rodrigues-Silva, R., Hulstijn, M., Neves, R.H., de Souza Panasco, M.,Lenzi, H.L., Machado-Silva, J.R., 2002. Natural Schistosoma mansoni infection inNectomys squamipes: histopathological and morphometric analysis incomparison to experimentally infected N. squamipes and C3H/He mice.Memorias do Instituto Oswaldo Cruz 97, 129–142.

Crane, F.L., 2001. Biochemical functions of coenzyme Q10. Journal of the AmericanCollege of Nutrition 20, 591–598.

Curtis, J., Minchella, D.J., 2004. Schistosome population genetic structure: whenclumping worms is not just splitting hairs. Parasitology Today 16, 68–71.

Daoud, A.A., Abdel-Ghaffar, A.E., Deyab, F.A., Essa, T.M., 2000. The effect ofantioxidant preparation (Antox) on the course and efficacy of treatment oftrichinosis. Journal of the Egyptian Society of Parasitology 30, 305–314.

Ding, A.H., Nathan, C.F., Stuehr, D.J., 1988. Release of reactive nitrogen intermediatesand reactive oxygen intermediates from peritoneal macrophages. Journal ofImmunology 141, 2407–2412.

El-Sokkary, G.H., Omar, H.M., Hassanein, A.M., Cuzzocera, S., Reiter, R.J., 2001.Melatonin reduces oxidative damage and increases survival of mice infectedwith Schistosoma mansoni. Free Radical Biology & Medicine 32, 319–332.

Failli, P., De Franco, R., Caligiuri, A., Romanelli, R.G., Marra, F., Batignani, G., Guerra,C.T., Laffi, G., Gentilini, P., Pinzani, M., 2000. Nitro-vasodilators inhibit platelet-derived growth factors induced-proliferation and migration of activated humanhepatic stellate cells. Gastroenterology 119, 479–492.

Ferré, N., Camps, J., Prats, E., Vilella, E., Paul, A., Figuera, L., 2002. Serum paraoxonaseactivity: a new additional test for the improved evaluation of chronic liverdamage. Clinical Chemistry 48, 261–266.

Ferro, D., Basili, S., Pratico, D., Iuliano, L., Fitzgerald, G.A., Violi, F., 1999. Vitamin Ereduces monocyte tissue factor expression in cirrhotic patients. Blood 93, 2945–2950.

Friedman, S.L., 2000. Molecular regulation of hepatic fibrosis, an integrated cellularresponse to tissue injury. Journal of Biological Chemistry 275, 2247–2250.

Gangadharan, R., Antrobus, R.A., Dwek, A., Zitzmann, M., 2007. Novel serumbiomarker candidates for liver fibrosis in hepatitis patients. Clinical Chemistry53, 1792–1799.

Gharib, B., Abdallahi, O.M., Dessein, H., de-Reggi, M., 1999. Development ofeosinophil peroxidase activity and concomitant alteration of the antioxidantdefenses in the liver of mice infected with Schistosoma mansoni. Journal ofHepatology 30, 594–602.

Gharib, B., Hanna, S., Abdallahi, O.M., Leidi, H., Gardette, B., De Reggi, M., 2001. Anti-inflammatory properties of molecular hydrogen: investigation on parasite-induced liver inflammation. CR Academie des Sciences III 324, 719–724.

Grisham, M.B., Jourd’Heuil, D., Wink, D.A., 1998. Nitric oxide I. Physiologicalchemistry of nitric oxide and its metabolites: implications in inflammation.American Journal of Physiology 278, 315–321.

Ismail, M., Metwally, A., Farghaly, A., Bruce, G., Tao, L., Bennet, G.L., 1996.Characterization of isolates of Schistosoma mansoni from Egyptian villagersthat tolerate high doses of praziquantel. American Journal of Tropical Medicineand Hygiene 55, 214–218.

Kaplowitz, N., 2000. Mechanisms of liver cell injury. Journal of Hepatology 32, 39–47.Kilic, S., Aydin, S., Kilic, N., Erman, F., Aydin, S., Celik, I., 2005. Serum arylesterase and

paraoxonase activity in patients with chronic hepatitis. World Journal ofgastroenterology 11, 7351–7354.

Lieber, C.S., 2000. Alcoholic liver disease: new insights in pathogenesis lead to newtreatments. Journal of Hepatology 32, 113–128.

Loguercio, C., Federico, A., 2003. Oxidative stress in viral and alcoholic hepatitis.Free Radical Biology & Medicine 34, 1–10.

Lorentz, K., Flatter, B., Augustin, E., 1979. Arylesterase in serum: elaboration andclinical application of a fixed-incubation method. Clinical Chemistry 10, 1714–1720.


Miles, M.V., Patterson, B.J., Chalfonte-Evans, M.L., Horn, P.S., Hickey, F.J., Schapiro,M.B., Steele, P.E., Tang, P.H., Hotze, S.L., 2007. Coenzyme Q10 (ubiquinol-10)supplementation improves oxidative imbalance in children with trisomy 21.Pediatric Neurology 37, 398–403.

Moron, M.S., Depierre, J.W., Mannervik, B., 1979. Levels of glutathione, glutathionereductase and glutathione-s-transferase activities in rat lung and liver.Biochimica Biophysica Acta 582, 67–78.

Ohkawa, H., Ohishi, N., Yagi, K., 1979. Assay for lipid peroxides in animal tissues bythiobarbituric acid reaction. Analytical Biochemistry 95, 351–358.

Parola, M., Robino, G., 2001. Oxidative stress-related molecules and liver fibrosis.Journal of Hepatology 35, 297–306.

Peters, P.A., Warren, K.S., 1969. A rapid method of infecting mice and otherlaboratory animals with S. mansoni: subcutaneous injection. Journal ofParasitology 55, 558.

Pietrangelo, A., Borella, F., Casalgrandi, G., Montosi, G., Ceccarelli, D., Gallesi, D.,Giovannini, F., Gasparetto, A., Masini, A., 1995. Antioxidant activity of sylibinin vivo during chronic iron overload in rat. Gastroenterology 109, 1941–1949.

Ruiz-Jimenez, J., Priego-Capote, F., Mata, J.M., Quesada, J.M., Luque de Castro, M.D.,2007. Determination of the ubiquinol-10 and ubiquinone-10 (coenzyme-Q10)in human serum by lipid chromatography tandem mass spectrometry toevaluate the oxidative stress. Journal of Chromatography A 1175, 242–248.

Salem, S.A., El-Sheikh, T.F., 1998. A preliminary study of the role of an antioxidantpreparation (Antox) on Trichinella spiralis infection in albino rats. EgyptianJournal of Medical Microbiology 7, 175–184.

Sangster, N.C., 2001. Managing parasiticide resistance. Veterinary Parasitology 98,89–109.

Sayed, H.A., EL-Ayyat, A., Kader, A.A., Sabry, H.Y., Amer, N.M., 2004. Epidemiology ofSchistosoma mansoni infection and its relationship to snail distribution in avillage at the Nile bank south to Cairo. Journal of the Egyptian Public HealthAssociation 79, 95–113.

Shoheib, Z.S., 2002. Study of the biological changes in experimental trichinellosis inrats under the influence of chemotherapy. M.D. Thesis. ParasitologyDepartment, Faculty of Medicine, Tanta University, Egypt.

Singh, R.B., Shinde, S.N., Shopoa, R.K., Niaz, M.A., Thakur, A.S., Onouchi, Z., 2000.Effect of coenzyme Q10 on experimental atherosclerosis and chemicalcomposition and quality of atheroma in rabbits. Atherosclerosis 148, 275–282.

Soltys, K., Dikdan, G., Koneru, P., 2001. Oxidative stress in fatty livers of obeseZucker rats: rapid amelioration and improved tolerance to warm ischemia withtocopherol. Hepatology 34, 13–18.

Stavitsky, A.B., 2004. Regulation of granulomatous inflammation in experimentalmodels of schistosomiasis. Infection and Immunity 72, 1–12.

Tsukamoto, H., Horne, W., Kamimura, S., Niemela, O., Parkkila, S., Ylä-Herttuala, S.,Brittenham, G.M., 1995. Experimental liver cirrhosis induced by alcohol andiron. Journal of Clinical Investigation 96, 620–630.

Turkodgan, M.K., Agaoglu, Z., Yener, Z., Sekeroglu, R., Akkan, H.A., Avci, M.E., 2001.The role of antioxidant vitamins (C and E), selenium, and Nigella sativa in theprevention of liver fibrosis and cirrhosis in rabbits: new hopes. DeutscheTierarztliche Wochenschrift 108, 71–73.

Wilkinson, J.H., Baron, D.N., Moss, D.W., Walker, P.G., 1972. Standardization ofclinical enzyme assays: a reference method for aspartate and alaninetransaminase. Journal of Clinical Pathology 25, 940–944.

Wilson, M.S., Mentink-Kane, M.M., Pesce, J.H., Ramalingam, T., Thompson, R., Wynn,T.A., 2007. Immunopathology of schistosomiasis. Immunology & Cell Biology85, 148–154.

Wynn, T.A., Thompson, R.W., Cheever, A.W., Mentink-Kane, M.M., 2004.Immunopathogenesis of schistosomiasis. Immunological Reviews 201, 156–167.

Yasuda, M., Shimizu, I., Shiba, M., Ito, S., 1999. Suppressive effects of estradiol ondimethyl-nitrosamine-induced fibrosis of the liver in rats. Hepatology 29, 719–727.

Zhen, M., Wang, Q., Huang, X., Cao, L., Chen, X., Sun, K., Liu, Y., Li, W., Zhang, L., 2007.Green tea polyphenol epigallocatechin-3-gallate inhibits oxidative damage andpreventive effects on carbon tetrachloride-induced hepatic fibrosis. Journal ofNutrition and Biochemistry 18, 795–805.

experimental parasitology - tanta...

Documents