Biochemical Evaluation of Antioxidant Enzyme Activities and Lipid Peroxidation Level Associated with Liver Enzymes in Patients with Fascioliasis.

Fascioliasis, which is caused by infection with Fasciola gigantica and Fasciola hepatica, is a zoonotic disease with a global distribution. This comparative study aimed to investigate antioxidant enzyme activities and oxidative status of chronic fascioliasis patients. In this study, 20 patients were compared with 10 controls and the levels of superoxide dismutase (SOD), malondialdehyde (MDA), glutathione peroxidase (GPX), catalase (CAT), alanine aminotransferase (ALT) and aspartate aminotransferase (AST) enzymes were evaluated. The results showed that MDA, CAT, AST, and ALT levels were higher in patients than in controls, while SOD and GPX levels were higher in controls than in patients (P≤0.05). Moreover, the enzyme MDA showed a positive correlation with SOD and GPX in the infected group. The enzyme SOD had an indirect correlation with CAT and a direct correlation with GPX. The positive correlation between ALT and AST was shown to be extremely significant (P≤0.05). The significant decrease in antioxidant enzymes and an increase in serum lipid peroxidation in the red blood cells of patients with fascioliasis indicated the presence of oxidative stress, which showed inflammation and oxidative stress, the pathogenesis of which was indicative of the stage of infection.

Oxidative status and changes in the adenosine deaminase activity in experimental host infected with tropical liver fluke, Fasciola gigantica.

Fine tuning of the metabolic, physiological and immunological cues along with interplay between the biomolecules of the host and the parasite could be responsible for the successful establishment of parasitic infections. The present investigation was aimed at evaluating the oxidative status and the level of adenosine deaminase (ADA) in the serum and liver of rabbits experimentally infected with Fasciola gigantica. A significant increase in level of ROS, MDA and 4-HNE along with a decline in the SOD, CAT, GR and GST activity was evident in rabbits experimentally infected with Fasciola gigantica. However, there was an increase in the GPX activity in the sera of infected rabbits. The increased GPX activity and decreased GR activity would have resulted in the depletion of GSH, a key non-enzymatic antioxidant, in the infected animals. The level of GSSG was also found to be higher in the sera and liver tissues of the infected rabbits along with a decline in the GSH/GSSG ratio, indicating a high level of oxidative stress in the infected animals, which also showed a significant increase in the activity of the marker enzymes of liver pathology, AST and ALT. Further, a significant inhibition of the adenosine deaminase (ADA) activity in the infected rabbits was accompanied with the reduction in the level of pro-inflammatory cytokine, IL-6 while the anti-inflammatory cytokine, IL-4 level was significantly elevated. In conclusion, the F. gigantica induced significant oxidative stress as evident from the increased levels of ROS and lipid peroxidation along with the disruption of antioxidant and detoxification cascade ultimately lead to pathogenic and inflammatory responses in the experimental host. Whereas, the altered ADA activity could modulate the host's immune responses toward Th-2 type and would facilitate the successful establishment of flukes within their host, thus indicating that ADA could be exploited as a target for the development of novel anthelmintic drugs against fasciolosis.

Dynamics of liver enzymes in rabbits experimentally infected with Fasciola sp. (Intermediate form from Japan).

Dynamics of serum liver enzymes in rabbits experimentally infected with metacercariae of Fasciola sp. (intermediate form between Fasciola hepatica and F. gigantica) were monitored. Gradual increase of serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were observed from 3 weeks post-inoculation (WPI) and peaked at 6 WPI, which corresponded well to the period of migration and development of juvenile fluke in the liver parenchyma and the time when the young adult flukes migrated to the bile duct. However, no significant increase in serum gamma-glutamyl transferase (GGT) and alkaline phosphatase (ALP) were observed. This could reflect reduced or minimal injury of bile ducts and biliary epithelia as the flukes had reached the adult stage. Alpha- fetoprotein (AFP) and carcinoembryonic antigen (CEA) were not detected in the infected rabbit during the course of the experiment. Serum liver enzymes monitoring might be useful for understanding the host-parasite relationship in fascioliasis.

Activities of ectonucleotidases and adenosine deaminase in platelets of cattle experimentally infected by Fasciola hepatica.

The enzymatic activities of NTPDase, 5'-nucleotidase and adenosine deaminase (ADA) are important in regulating the concentration of adenine nucleotides, molecules known to be involved on platelet aggregation. Fasciolosis causes coagulation disorders that have not been completely elucidated. Taking into consideration the association between the purinergic system and hemostasis, this study aimed to evaluate the enzymatic activities of NTPDase (hydrolyze ATP and ADP), 5'-nucleotidase (hydrolyze AMP) and ADA (deamination of adenosine) in platelets from cattle experimentally infected by Fasciola hepatica on days 20, 40, 60 and 80 post-infection (PI). For this study, 10 healthy Friesian steers were separated into two groups: the group A (n = 5) was used as uninfected control, and the group B was composed of steers experimentally infected by F. hepatica (n = 5). The number of platelets did not differ between groups in the periods evaluated. Reduction of NTPDase (p < 0.05) hydrolysing ATP (days 20, 40 and 60 PI), and ADP (days 40, 60 and 80 PI), and on 5'-nucleotidase hydrolyzing AMP (days 40 and 60 PI) was observed. A reduction (p < 0.05) in ADA activity on day 20 PI, as well as an increase (p < 0.05) in ADA activity on days 40 and 60 PI was observed when compared to the control. Based on these results, we can conclude that ATP, ADP and AMP hydrolysis and adenosine deamination were altered in platelets of cattle infected by F. hepatica. Considering the importance of the purinergic system in hemostasis, it is believed that those changes may contribute to the coagulation impairment observed in acute fasciolosis described in the literature.

Oxidative stress and changes in adenosine deaminase activity of cattle experimentally infected by Fasciola hepatica.

The aim of this study was to evaluate the oxidative stress in serum and liver and adenosine deaminase (ADA) activity of cattle experimentally infected by Fasciola hepatica. The group A consisted of five healthy animals (uninfected), and the group B was composed of five animals orally infected with 200 metacercariae of F. hepatica. On days 20, 40, 60 and 80 post-infection (PI) serum was collected to measure oxidative stress variables. On day 100 PI, animals were humanely euthanized and liver samples were collected. Infected animals showed lower (P < 0·05) seric ADA activities on days 40 and 60 PI but higher (P < 0·05) in the liver tissue compared with uninfected animals. Seric and hepatic reactive oxygen species (ROS) were higher (P < 0·05) in infected compared with uninfected animals. Hepatic thiobarbituric acid reactive substances were higher (P < 0·05) in infected animals. Catalase and glutathione S-transferase activities were lower in liver tissue of infected animals, while glutathione peroxidase was higher compared with uninfected (P < 0·05). In summary, we conclude that oxidative stress occurs in cattle experimentally infected by F. hepatica, mainly due to excessive ROS production in the course of fasciolosis, contributing to hepatic damage, and that increased in hepatic ADA activity may contribute to the inflammatory process.

Seric and hepatic NTPDase and 5' nucleotidase activities of rats experimentally infected by Fasciola hepatica.

The enzymatic activities of NTPDase and 5'nucleotidase are important to regulate the concentration of adenine nucleotides, known molecules involved in many physiological functions. Therefore, the objective of this study was to evaluate the activity of NTPDase and 5'nucleotidase in serum and liver tissue of rats infected by Fasciola hepatica. Rats were divided into two groups: uninfected control and infected. NTPDase activity for adenosine triphosphate (ATP) and ADP substrates in the liver was higher compared with the control group at 15 days post-infection (PI), while seric activity was lower. In addition, seric and hepatic samples did not show changes for 5'nucleotidase activity at this time. On the other hand, either NTPDase or 5'nucleotidase activities in liver homogenate and serum were higher at 87 days PI. Early in the infection, low NTPDase activity maintains an increase of ATP in the bloodstream in order to activate host immune response, while in hepatic tissue it decreases extracellular ATP to maintain a low inflammatory response in the tissue. As stated, higher NTPDase and 5'nucleotidase activities 87 days after infection in serum and tissue, probably results on an increased concentration of adenosine molecule which stimulates a Th2 immune response. Thus, it is possible to conclude that F. hepatica infections lead to different levels of nucleotide degradation when considering the two stages of infection studied, which influences the inflammatory and pathological processes developed by the purinergic system.

Relation between iron metabolism and antioxidants enzymes and δ-ALA-D activity in rats experimentally infected by Fasciola hepatica.

The aim of this study was to evaluate the iron metabolism in serum, as well as antioxidant enzymes, in addition to the Delta-Aminolevulinic Acid Dehydratase (δ-ALA-D) activity in the liver of rats experimentally infected by Fasciola hepatica. Thirty male adult rats (Wistar) specific pathogen free were divided into four groups: two uninfected group (CTRL 1 and CTRL 2) with five animals each and two infected groups (INF 1 and INF 2) with 10 animals each. Infection was performed orally with 20 metacercariae at day 1. On day 15 (CTRL 1 and INF 1 groups) and 87 PI (CTRL 2 and INF 2 groups) blood and bone marrow were collected and the animals were subsequently euthanized for liver sampling. Blood was allocated in tubes without anticoagulant for serum acquisition to measure iron, transferrin and unsaturated iron binding capacity (UIBC). δ-ALA-D, superoxide dismutase (SOD), and catalase (CAT) activities were measured in the liver. A decrease in iron, transferrin and UIBC levels was observed in all infected animals compared to control groups (P < 0.05). Furthermore, iron accumulation was observed in bone marrow of infected mice. Infected animals showed an increase in δ-ALA-D activity at 87 post-infection (PI) (INF 2) as well as in SOD activity at days 15 (INF 1) and 87 PI (INF 2). On the other hand, CAT activity was reduced in rats infected by F. hepatica during acute and chronic phase of fasciolosis (INF 1 and INF 2 groups), when moderate (acute) and severe necrosis in the liver histopathology were observed. These results may suggest that oxidative damage to tissues along with antioxidant mechanisms might have taken part in fasciolosis pathogenesis and are also involved in iron deficiency associated to changes in δ-ALA-D activity during chronic phase of disease.

Activity of cholinesterases, pyruvate kinase and adenosine deaminase in rats experimentally infected by Fasciola hepatica: Influences of these enzymes on inflammatory response and pathological findings.

The aim of this study was to investigate acetylcholinesterase (AChE) in total blood and liver tissue; butyrylcholinesterase (BChE) in serum and liver tissue; adenosine deaminase (ADA) in serum and liver tissue; and pyruvate kinase (PK) in liver tissue of rats experimentally infected by Fasciola hepatica. Animals were divided into two groups with 12 animals each, as follows: group A (uninfected) and group B (infected). Samples were collected at 20 (A1 and B1;n=6 each) and 150 (A2 and B2; n=6 each) days post-infection (PI). Infected animals showed an increase in AChE activity in whole blood and a decrease in AChE activity in liver homogenates (P<0.05) at 20 and 150 days PI. BChE and PK activities were decreased (P<0.05) in serum and liver homogenates of infected animals at 150 days PI. ADA activity was decreased in serum at 20 and 150 days PI, while in liver homogenates it was only decreased at 150 days PI (P<0.05). Aspartate aminotransferase and alanine aminotransferase activities in serum were increased (P<0.05), while concentrations of total protein and albumin were decreased (P<0.05) when compared to control. The histological analysis revealed fibrous perihepatitis and necrosis. Therefore, we conclude that the liver fluke is associated with cholinergic and purinergic dysfunctions, which in turn may influence the pathogenesis of the disease.

Increase of gluthatione S-transferase, carboxyl esterase and carbonyl reductase in Fasciola hepatica recovered from triclabendazole treated sheep.

Fasciolasis is a zoonotic parasitic disease caused by Fasciola hepatica and its control is mainly based on the use of triclabendazole (TCBZ). Parasite resistance to different anthelmintics is growing worldwide, including the resistance of F. hepatica to TCBZ. In the present work we evaluate "in vivo" the activity of xenobiotic metabolizing enzymes of phase I (carboxyl esterases) and phase II (glutathione S-transferases and carbonyl reductases) recovered of flukes from sheep treated with TCBZ. All three enzymes showed increased activity in TCBZ flukes returning 60h post-treatment at similar to baseline unexposed flukes. TCBZ action may induce secondary oxidative stress, which may explain the observed increment in activities of the analyzed enzymes as a defensive mechanism. The enzymes analyzed are candidates to participate actively in the development of resistance at TCBZ in F. hepatica.

Resistance of Fasciola hepatica against triclabendazole in cattle in Patagonia (Argentina).

In the winter of 2008, cattle on a farm in the province of Neuquen, Argentina died from subacute and chronic liver fluke disease despite four previous treatments with Triclabendazole (TCBZ). In the spring of 2009, a preliminary efficacy test revealed good performance using nitroxynil, whereas TCBZ efficacy was only 18% by egg counts of Fasciola eggs in the faeces. Resistance to Fasciola hepatica to TCBZ has never been reported in South America, so in January of 2010 a controlled trial was conducted to confirm and to define the degree of resistance in this herd. In a clinical trial, the fluke egg output was monitored on Days 14 and 21 and serum enzymes gamma-glutamyl transpeptidase (GGT) and glutamic-oxaloacetic transaminase (GOT) on Days 0 and 21 in 36 calves treated with TCBZ or with closantel. The results showed a reduction of 100% in fluke egg output at Days 14 and 21 for closantel. The mean epg in the TCBZ-treated groups did not decrease. Because of the fact that in this study TCBZ treatment in cattle had no effect, even at double the recommended dose, it is highly indicative that resistance of F. hepatica against TCBZ is present on this farm. The GGT and GOT levels decreased in the closantel-treated group as a result of the treatment at 21 days after dosing. To evaluate the importance of TCBZ resistance in F. hepatica in Argentina, a study on more farms from endemic areas is needed.

Helminth 2-Cys peroxiredoxin drives Th2 responses through a mechanism involving alternatively activated macrophages.

During helminth infections, alternatively activated macrophages (AAMacs) are key to promoting Th2 responses and suppressing Th1-driven inflammatory pathology. Th2 cytokines IL-4 and/or IL-13 are believed to be important in the induction and activation of AAMacs. Using murine models for the helminth infections caused by Fasciola hepatica (Fh) and Schistosoma mansoni (Sm), we show that a secreted antioxidant, peroxiredoxin (Prx), induces alternative activation of macrophages. These activated, Ym1-expressing macrophages enhanced the secretion of IL-4, IL-5, and IL-13 from naive CD4(+) T cells. Administration of recombinant FhPrx and SmPrx to wild-type and IL-4(-/-) and IL-13(-/-) mice induced the production of AAMacs. In addition, Prx stimulated the expression of markers of AAMacs (particularly, Ym1) in vitro, and therefore can act independently of IL-4/IL-13 signaling. The immunomodulatory property of Prx is not due to its antioxidant activity, as an inactive recombinant variant with active site Cys residues replaced by Gly could also induce AAMacs and Th2 responses. Immunization of mice with recombinant Prx or passive transfer of anti-Prx antibodies prior to infection with Fh not only blocked the induction of AAMacs but also the development of parasite-specific Th2 responses. We propose that Prx activates macrophages as an initial step in the induction of Th2 responses by helminth parasites and is thereby a novel pathogen-associated molecular pattern.

Fascioliasis in pregnancy.

BACKGROUND: Fascioliasis is a common zoonotic infection worldwide, although cases in the United States are uncommon, sporadic, and predominantly found in the immigrant population. The small number of cases identified in the United States may reflect the unfamiliarity of physicians with this infection. CASE: A 28-year-old Hispanic woman who frequently visited northern Central Mexico presented at 36 weeks of gestation with nausea, vomiting, and right upper quadrant pain. She was diagnosed with cholelithiasis. Postpartum endoscopic retrograde cholangiopancreatography and sphincterotomy were performed, with discovery of the trematode Fasciola hepatica. The patient received triclabendazole, which led to clinical improvement. CONCLUSION: Fascioliasis often mimics another common problem in pregnancy, cholelithiasis; clinicians need to be aware of this disease in high-risk populations.

The silencing of cysteine proteases in Fasciola hepatica newly excysted juveniles using RNA interference reduces gut penetration.

Probing protein function in parasitic flatworms is hampered by the difficulties associated with the development of transgenic approaches. Although RNA interference (RNAi) in schistosomes shows much promise, it has not been reported in other trematodes. Here, we show the successful silencing of the cysteine proteases cathepsin B and L in the infective stage of Fasciola hepatica newly excysted juveniles (NEJs). Silencing resulted in marked reductions in target transcript levels and significant diminution in the encoded proteins in the gut. RNAi of either enzyme in NEJs induced transient, abnormal locomotory phenotypes, and significantly reduced penetration of the rat intestinal wall.

Lipid peroxidation level and antioxidant enzyme activities in the blood of patients with acute and chronic fascioliasis.

OBJECTIVE: In this study, we investigated the relationship between fascioliasis and serum malondialdehyde (MDA) levels, superoxide dismutase (SOD), catalase, and glutathione peroxidase (GPx) activities. We also investigated whether there are significant differences in MDA levels and antioxidant enzymatic activities between acute and chronic fascioliasis. METHODS: Forty fascioliasis patients who were diagnosed by ES-ELISA positivity were included in this study. The patients were classified as 18 with acute and 22 with chronic fascioliasis. RESULTS: In patients with fascioliasis, levels of MDA were statistically higher and erythrocyte SOD and GPx activities were statistically lower than in healthy controls. MDA levels were found to be higher in patients with acute fascioliasis than in patients with chronic fascioliasis although MDA levels were significantly higher in patients with chronic fascioliasis than in controls. There was no statistically significant difference between the two groups for the antioxidant enzyme activities. CONCLUSION: The results of this study may indicate that fascioliasis produces specific effects on the antioxidant defense mechanisms due to its inflammatory character. Our results also allow us to suggest that oxidative stress has an important role in the pathogenesis of fascioliasis and the persistence of this oxidative stress can be one of the underlying factors in the pathogenesis of the chronic disease.

The relationship between haematological indices, serum gamma-glutamyl transferase and glutamate dehydrogenase, visual hepatic damage and worm burden in cattle infected with Fasciola gigantica.

The association between visual hepatic damage, burden of Fasciola gigantica, serum levels of gamma glutamyl transferase (GGT) and glutamate dehydrogenase (GLDH) is described from an abattoir study of 70 cattle in the Philippines. In another abattoir study of 60 cattle, the relationship between burden of F. gigantica and haematological indices was investigated. The degree of visual hepatic damage and burden of F. gigantica were significantly positively related to levels of GGT and GLDH. Red blood cell counts and packed cell volume were significantly inversely related to worm burden, but animals compensated for reduced numbers of red blood cells by increasing red cell haemoglobin content.

Serological changes in goats experimentally infected with Fasciola gigantica in Buea sub-division of S.W.P. Cameroon.

Based on the recent increase in the rearing of West African Dwarf (WAD) goats in this environment, a study of biochemical parameters was carried out in healthy and infected goats to obtain baseline data for monitoring the health and productive indices in breeding programmes for the goats. The results revealed variations in some of the parameters studied. The serum minerals potassium, chlorine and bicarbonate did not show any significant differences (P>0.05) between the infected and control groups of goats, but there was a significant difference (P<0.0001) in sodium, calcium and urea levels. For serum lipids cholesterol and triglyceride, there were significant (P<0.0001) differences between the infected and control group. This goes same for serum proteins, albumin and total proteins. However, serum enzymes, serum glutamic oxaloacetic transaminase (SGOT) and gamma glutamyl transferase (GGT) were not significantly different (P>0.05) between the infected and control groups. There was a significant difference (P<0.0001) in alkaline phosphatase (ALP) and serum glutamic pyruvic transaminase (SGPT) between the infected and control groups.

[Some enzymes associated with lipid catabolism in the Fasciola hepatica larvae--Galba truncatula system. I. Esterases and lipases]. / Niektóre enzymy zwiqzane z rozkladem lipidów w ukladzie: stadia rozwojowe Fasciola hepatica--Galba truncatula. I. Esterazy i lipazy.

Histochemical and cytofotometric techniques were used to detect esterase and lipase in the hepatopancreas of the snail Lymnaea truncatula and in larval stages of its parasite Fasciola hepatica. Activities of both enzymes were shown to increase in the infected hepatopancreas, compared with the control. The effect is ascribed by the authors to compensation processes emerging as a defence mechanism in the snail. Both enzymes, esterase and lipase proved to be highly active in tissues of sporocysts, rediae and cercariae, thus demonstrating the larvae to be capable of katabolism of lipids and utilising them as an energy source.

Diagnosis of parasitic zoonoses by immunoenzymatic assays--analysis of cross-reactivity among the excretory/secretory antigens of Fasciola hepatica, Toxocara canis, and Ascaris suum.

Several parasitic infections such fasciolosis, toxocariosis or ascariosis are important zoonoses. During the infection with Fasciola hepatica, Toxocara canis and Ascaris suum, an important intraorganic phase in their hosts takes place, releasing antigens responsible for a humoral immune response, which enables the diagnosis of that parasitosis. A study to identify the existence of cross-reactivity among the excretory/ secretory antigens of F. hepatica, T. canis and A. suum was developed. One group of Sprague-Dawley rats was infected with 20 metacercariae of F. hepatica and another group remained uninfected as control. By means of an Indirect-ELISA, the rat humoral immune response (IgG and IgM) against the excretory/secretory antigens of F. hepatica was analysed and measured for cross reactivity with T. canis and A. suum. IgM cross-reaction was mainly observed in the first 10 weeks post-infection. IgG cross-reaction was observed throughout the study, and was maximal at the 2-3 weeks and 3-6 weeks post-infection, which corresponds to the intraorganic migratory phase of these parasites. The western-blot showed that the rat IgG recognised three proteins of 190, 160 and 33 kDa in the antigens from F. hepatica, T. canis and A. suum. The existence of cross-reactivity among these antigens seems to demonstrate also the presence of structural similarities, such as tegumental proteins. These results should be consider when immunoassay probes are used in the diagnosis of parasitic infections.

Comparison of hepatic and renal drug-metabolising enzyme activities in sheep given single or two-fold challenge infections with Fasciola hepatica.

The activity of drug-metabolising enzymes was compared in liver and kidneys of adult sheep given single or two-fold fluke infection. Fascioliasis was induced by oral administration of 200 metacercariae of Fasciola hepatica to female sheep either 10 or 20 weeks (mono-infections) or 10 and 20 weeks (bi-infection) before killing. The parasitic pathology was ascertained at autopsy and by clinical observation of animals. In the liver of both mono- and bi-infected animals, significant decreases (P<0.05) (17-44%) were observed in the microsomal content of cytochrome P450 and in the two measured P450-dependent monooxygenase activities, benzphetamine and ethylmorphine N-demethylations. Moreover, Western blot analysis of microsomes demonstrated a decrease in the expression of cytochrome P4503A subfamily correlative with that of its presumed corresponding activity ethylmorphine N-demethylase. By contrast, the conjugation of chloro-dinitrobenzene to glutathione remained unchanged in liver cytosolic fractions prepared from all these animals. In kidneys, a significant decrease (P<0.05) (30%) in microsomal cytochrome P450 level of 10-week mono-infected sheep was observed whereas there was no change in the other groups of animals. The inflammatory origin and the consequences in terms of pathology and animal productivity of the fascioliasis-induced decreases in tissue-oxidative drug metabolism are discussed, particularly in the case of adult sheep suffering repetitive infections.

Induced expression of CYP2A5 in inflamed trematode-infested mouse liver.

Trematode infections have long been associated with specific types of cancer. We investigated the ability of the liver fluke Fasciola hepatica to alter host enzymes in a manner that might provide insight into the phenomenon of biologically associated cancers. Our data demonstrate an increased activity of the CYP2A5 isozyme in male mouse liver infected with F.hepatica. Induction of this enzyme was further assessed immunohistochemically. The infection affected CYP2A5 distribution in hepatic tissue. Inflammation and proliferation in liver tissue were observed at the same time that CYP2A5 activity increased. This enzyme is known to participate in the metabolism of several carcinogens which are common contaminants in environments of developing countries where parasitic infections may be prevalent.