Differential Activities of Glutathione S-Transferase Isoenzymes in Strains of Fasciola Hepatica Susceptible and Resistant to Triclabendazole

Corresponding Author: V. Fernández Laboratorio de Biología Celular y Molecular. Centro de Investigación Veterinaria de Tandil, Argentina Email: vanesaf@vet.unicen.edu.ar Abstract: Fasciolosis, a parasitic zoonosis of intrahepatic location, is caused by the trematode Fasciola hepatica. Its control is mainly based on the use of the anthelminthic Triclabendazole (TCBZ). The indiscriminate use of this drug has favored the development of anthelmintic resistance. The Glutation S-Transferases (GSTs) are multifunctional enzymes involved in the detoxification of xenobiotics and endogenous compounds using conjugation with endogenous glutathione. Recently, it has been shown an active participation of this family of enzymes in the detoxification of TCBZ related to the phenomenon of resistance. In F. hepatica, eight isoenzymes of the GST are present. Since it is well known that different isoenzymes do not necessarily have the same metabolic activity, this study evaluated the cytosolic activity of mu and pi GST isoenzymes in TCBZ resistant (Sligo and Oberon strains) and TCBZ susceptible (Cullompton strains) of F. hepatica. The results obtained in this study confirm that, although both isoenzymes are involved in different processes of detoxification in F. hepatica, only the GSTmu isoenzyme is involved in the manifestation of resistance to TCBZ.


Introduction
Fasciolosis is a parasitic zoonotic disease caused by the trematode Fasciola hepatica, a parasite of ruminants reared in temperate regions of the world. This parasitic disease is of great economic importance because annual losses due to this infection are estimated at more than $ 3 billion annually (Boray, 1994). In addition, fasciolosis has become an emerging zoonosis in many countries (Mas-Coma et al., 2009a) estimating that 17 million people are currently infected and 180 million are at risk of infection (WHO, 2007). In Europe, in recent years an increase in cases of cattle fasciolosis have been reported due to meteorological changes that determine a different distribution of the snail Galba truncatula, that it is the intermediate host required (Mas-Coma et al., 2009b).
The control of fasciolosis is mainly based on the use of the anthelmintic Triclabendazole (TCBZ), a halogenated benzimidazole which shows excellent efficacy against the juvenile and adult stages. On the other hand, parasites activate the induction of certain metabolic enzymes that deactivate Xenobiotics (XME) like anthelmintics, thus facilitating the survival of the exposed helminth (Brennan et al., 2007). This metabolic response to anthelmintics can initiate the phenomenon of resistance including resistance of F. hepatica to TCBZ. This ability to deactivate anthelmintics through biotransformation processes represents an advantageous defense strategy of parasites (Robinson et al., 2004). In parasitic helminths the Glutathione S-Transferases (GSTs) are the main phase II detoxification system. In F. hepatica, GST enzyme represents 4% of the total soluble protein distributed in various tissues with important physiological functions (Chemale et al., 2006).
The increase in the activity of enzymes such as Flavin monooxygenase (XME phase I) (Alvarez et al., 2005) and GST (XME phase II) (Scarcella et al., 2012) in TCBZ resistant trematodes (Sligo strain) provides an interesting contribution to the understanding of the phenomenon of resistance that these worms possess. This overexpression confirms that more than one metabolic pathway may be involved in the phenomenon of resistance against TCBZ (Scarcella et al., 2012). In F. hepatica, eight GST isoforms have been reported (Wijffels et al., 1992), some associated to cell membranes (mGST) that structurally and functionally is distinct from the cytosolic homotrimeric GST (cGST) (Mannervik and Widersten, 1995). Therefore the mGST differs from the cGSTs (Morgenstern et al., 1982) in the fact that can activate by-sulphydric reagents, such as Nethylmaleimide. This property is important to establish that it is not contaminated with cGST (Andersson et al., 1994). Most of the "in vitro" or "ex vivo" studies on the metabolic response of F. hepatica against TCBZ have shown a significant decrease in the activity of superoxide dismutase and an increase in the level of GST in both young and adult flukes after incubation with TCBZ sulphoxide (Shehab et al., 2009). Therefore, the interactions with these enzymatic systems can drastically affect the kinetic disposition of these drugs.
The present work evaluated the activity "in vitro" of total cGST and cGSTmu and cGSTpi isoenzymes in the susceptible (Cullompton) and resistant (Sligo and Oberon) strains of F. hepatica.

Material and Methods
Fifteen (15) weaned Corriedale lambs free of parasites were infected orally with 200 metacercariae of F. hepatica contained in a gelatin capsule. Five animals were infected with a F. hepatica susceptible to TCBZ (Cullompton strain) and five with the resistant strains respectively (Sligo and Oberon strains). For more details on the history of the three isolates see corresponding work (Robinson et al., 2004). Intrahepatic infection was confirmed 16 weeks later by the presence of eggs in feces and the indirect estimation of liver damage was determined by measuring the levels of the serum enzymes GLDH and GT (Fig. 3).
The animals were sacrificed by procedures and management protocols approved by the Ethics Committee in accordance with the policy of Animal Welfare (law 087/02) of the Faculty of Veterinary Medicine of the National University of the Center of the Province of Buenos Aires (UNCPBA), Tandil, Argentina (http://www.vet.unicen.edu.ar) (AVMA, 2001). The adult flukes were collected of the bile ducts and liver and subsequently processed to obtain the Cytosolic fractions (Cyt). The test conditions were as described elsewhere (Solana et al., 2009).
Fasciola hepatica (10 g) were washed with cold KCl (1.15%) and then transported to the laboratory covered with Phosphate Buffer Saline (PBS) (0.1 M, pH 7.4) at 4°C. All subsequent operations were between 0 and 4°C.
Each specimen was cut into small pieces and washed several times with PBS. The samples were homogenized (1:1) in PBS with a Homogenizer Ultra-Turrax (IKA Works Inc., Wilmington, USA), centrifuged at 10,000 g for 20 m and the resulting supernatant centrifuged at 100,000 g for 60 min. The supernatants of ultracentrifugation (Cyt) were collected and then stored at -80°C until subsequent analysis. The protein content of the Cyt fractions was determined using bovine serum albumin as standard (Lowry et al., 1951). The total Cytosolic GST (cGST) activity was determined using 1chloro, 2, 4-Dinitrobenzene (CDNB) as substrate. Ethacrynic acid was used as substrate to measure the GST-Pi activity and 1,4-Dichlorobenzene (DCNB) to measure GST-Mu activity. All these procedures were supervised by the continuous Spectrophotometric method (Habig et al., 1974).
Data on the activity of total cGST, GST-Mu and GST-Pi in the Cytosolic protein fraction (n = 13) of the trematodes were compared statistically using an Analysis of Variance (ANOVA). Statistical comparisons were performed using a two way ANOVA using the Bonferroni analysis by the software GraphPad INSTAT 3.00 (Graph Pad Software, Inc.,). Differences with p≤0,05 were considered significant.

Results and Discussion
The cytosolic fractions of F. hepatica resistant to TCBZ (Sligo and Oberon strains) expressed greater metabolic activity of cGST with respect to the susceptible strain (Cullompton) ( Table 1). The total activity of GST (n = 13) in Sligo and Oberon resistant strains was 59 and 52% respectively higher (p<0.001) than in the susceptible Cullompton strain.
When were analyzed the different isozymes are found different enzymatic activities. While the GST-Pi activity (n=13) did not differ between the different F. hepatica strains tested (Fig. 1), the GST-Mu activity had significant differences (Fig. 2).
The GST-Mu activity (n = 13) in the resistant Oberon strain was 1.37 nmol/min/mg of protein and in the Sligo strain was 1.28 nmol/min/mg of protein compared to the susceptible Cullompton strain (0.7 nmol/min/mg of protein) (Fig. 2).
This activity was 71 and 86% higher in the resistant strains compared to the susceptible strain ( Table 2). Confirming that this isoenzyme is actively involved in the mechanisms of TCBZ detoxification.

Conclusion
The increased activity of the total cGST (XEM Phase 2) in both strains TCBZ-R (Sligo and Oberon) observed in this study provides an understanding of the phenomenon of resistance and adds information to the knowledge of the response that the parasites have exposure to different xenobiotic. While the GST-Pi activity (substrate: Ethacrynic acid) did not differ between the different strains tested confirms its nonparticipation in the phenomenon of resistance to TCBZ, for another hand the isoenzyme GST-Mu (substrate: DCNB) has respectively 71 and 86% higher activity in the TCBZ-R strains (Sligo and Oberon) confirming that this isoenzyme is actively involved in the expression of increased metabolic activity. These results contribute to the understanding of this pathway and too add information at the knowing over the parasites and their answer to different xenobiotics.

Acknowledgement
The metacercariae of F. hepatica were kindly provided by Professor I. Fairweather, School of Biology and Biochemistry, The Queens University of Belfast, United Kingdom, Northern Ireland.

Funding Information
H. Solana is Adjunct Professional from the Comisión de Investigaciones Científicas (CIC-BA) de la Provincia de Buenos Aires, Argentina.

Ethics
The animals were sacrificed by procedures and management protocols approved by the Ethics Committee in accordance with the policy of Animal Welfare (law 087/02) of the Faculty of Veterinary Medicine of the National University of the Center of the Province of Buenos Aires (UNCPBA), Tandil, Argentina (http://www.vet.unicen.edu.ar ) (AVMA, 2001).