Fasciolahepatica is a helminth parasite that causes fascioliasis in domestic ruminants and humans. Economic losses due to its infection are estimated in US$ 2000–3000 million yearly . The anthelmintics are at present the only weapon against these parasitic helminths . The parasite resistance to different anthelmintics including that of F. hepatica to triclabendazole (TCBZ) is growing worldwide. Glutathione S‐transferases (GSTs) are enzymes involved in the detoxification of a wide range of substrates through chemical conjugation with glutathione, so that the product becomes more soluble in water, less toxic and easier to excrete. Eight GST isoenzymes are present in F. hepatica . Since the different isoenzymes do not necessarily have the same metabolic activity, in the present work, we evaluated the metabolic activity of total cytosolic GST and GST mu and GST pi isoenzymes in adult strains of F. hepatica susceptible (Cullompton) and resistant (Sligo and Oberon) to TCBZ of the highest metabolic activity of total GST. The genetic sequence database at the National Center for Biotechnical Information (NCBI) (GenBank ID: KF680281–KF680282) corresponding to the GST mu gene isolated from Cullompton strain (TCBZ‐susceptible) and (GenBank ID: KF680283–KF680284) corresponding to the GST mu gene isolated from Sligo strain (TCBZ‐resistant) in F. hepatica. Comparative analysis of both strains, Cullompton and Sligo, showed two nucleotide changes and change of one amino acid in the GST mu isoenzyme of the TCBZ‐resistant strain. These results together with the higher enzymatic activity of GST have a potential relevance as it contribute to the understanding the mechanisms that generate resistance to anthelmintics and the activity, metabolism, and disposition of these drugs in the parasite.
- Fasciola hepatica
- glutathione S-transferases
In fasciolosis, anthelmintic control is based mainly on the use of TCBZ, a halogenated benzimidazole thiol derivative that shows excellent efficacy against both juvenile (immature) and adult stages. The nematodicidal action of benzimidazoles (BZDs) is based on their binding to beta‐Tubulin, which produces subsequent disruption of the tubulin‐microtubule dynamic equilibrium . When the
The development of drug resistance can be facilitated by the action of xenobiotic metabolizing enzymes (XMEs) of phase I and phase II of detoxification . In all organisms, XMEs serve as an efficient defense against the potential negative action of xenobiotics. Several phase I enzymes are expressed in mammalians, where they introduce or unmask new functionalities on xenobiotic compounds. Examples of these enzymes include cytochrome P450s (Cyt P450), flavin‐containing monooxygenases (FMO), alcohol and aldehyde dehydrogenases, and esterases. To eliminate a large array of chemicals, living organisms have developed, in virtually all tissue enzyme systems, XMEs that transform exogenous and endogenous compounds into more hydrophilic derivatives through reactions collectively known as biotransformation. At present, much less is known about the activity of certain phase II enzymes and relatively less attention has been paid to hydrolytic and conjugative pathways. Many phase II reactions in mammals involve conjugating potentially toxic substances to glutathione. These reactions are mediated by the enzyme glutathione S‐transferase (GST) enzymes . Recent research has highlighted the importance of these transferases in the establishment of chronic helminth infections. These proteins appear to be the main phase II detoxification system present in parasitic worms. General biological roles of helminth GSTs include xenobiotic detoxification and ligand binding/transport functions .
As many as eight GST isoforms have been shown to be present in
Whereas the background about the interactions with such enzymatic systems may drastically affect the disposition kinetics of different drugs , the aim of the present work was to evaluate
2. Materials and methods
2.1. Collection of parasite material
Nine (9) parasite‐free Corriedale weaned lambs were orally infected each with 200 metacercariae of
2.2. Collection and processing of adult flukes
Adult flukes were collected from bile ducts and liver and processed. The collection of the flukes, their processing to obtain the cytosolic .
2.3. Preparation of cytosolic fractions
Parasite specimens (10–15 g) of the TCBZ‐susceptible or TCBZ‐resistant isolates of
The supernatant was collected and stored at −80°C until the analysis. Protein content from the supernatant fractions was determined using bovine serum albumin as a standard .
Total cGST activity using 1‐chloro, 2,4‐dinitrobenzene as substrate (CDNB), GST‐pi activity using ethacrynic acid as substrate and GST mu activity using 3,4‐dichloronitrobenzene (DCNB) as substrate were monitored by a continuous spectrophotometric method .
2.4. Reverse transcription polymerase chain reaction (RT‐PCR)
Total RNA was isolated from each strain of adult trematodes (
2.5. PCR amplification of cDNA
The PCR product was analyzed by electrophoresis in 1% agarose gel.
Total GST activity (
In the absence of an efficacious vaccine, chemotherapy remains the main tool in treating fasciolosis. Although other alternatives exist, current measures to control fasciolosis are based on the use of drugs such as triclabendazole (TCBZ) [29, 30].
Parasite defense mechanisms include detoxifying and anti‐oxidant enzymes that would suppress its oxidative killing . Therefore, it is necessary to know the mechanisms of detoxification and mechanism of anthelmintic resistance of
Other possibilities include enhanced substrate affinity of the enzymes brought about by mutations within their encoding genes. Residue changes may also influence the substrate specificity of the enzymes and could explain why TCBZ‐resistant flukes remain susceptible to ABZ [14, 32–35].
GSTs are regulated by a structurally diverse range of xenobiotics, and at least 100 chemicals have been identified to induce GSTs. Many of the compounds that induce GSTs are themselves substrates for these enzymes or are metabolized (by CytP450 or FMO) to compounds that can serve as GST substrates, suggesting that GST induction represents part of an adaptive response mechanism to chemical stress caused by electrophiles .
In the present work, cGST was analyzed in three strains of
The activity of
GST activity has this great potential importance as it might contribute to generating the phenomenon of resistance to TCBZ. These results contribute to the understanding not only of this metabolic pathway but also of the mechanism of resistance to TCBZ in
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