Increase Of Carboxylesterase Activity In Fasciola hepatica Recovered From Triclabendazole Treated Sheep

SCARCELLA S.; ORTIZ OBLITAS P.; LAMENZA P ; CEBALLOS L.; SOLANA H.

Congreso; 12th International Congress of the European Association for Veterinary Pharmacology and Toxicology (EAVPT); 2012

Fasciolasis is a zoonotic disease caused by the trematode Fasciola hepatica (Aldrige 1989). Treatment of liver fluke infested livestock is mainly based on the use of triclabendazole (TCBZ), an halogenated benzimidazole thiol derivative which showed efficiency against juvenile and adult stages, but recently has been compromised by emerging fluke resistance against this drug. Carboxylesterases (CE) are xenobiotic metabolizing enzymes (XMEs) that catalyze phase I biotransformation, producing  hydrolytic reactions of carboxylic ester, phosphate esters, amides and thioesters among other chemical compounds, playing an important role in endobiotic and xenobiotic metabolism in metazoans (Gregus and Klaassen 1996). In the present work, we evaluate in vivo the activity of carboxylesterase of F. hepatica exposed to Triclabendazole.  Four (4) parasite-free Corriedale weaned lambs were orally inoculated with 200 metacercariae of F. hepatica TCBZ-susceptible isolate (Strain Cullompton), contained in a gelatin capsule. The infection was confirmed 16 weeks later by the presence of eggs in feces and indirect estimation of liver damage in the host. The animals were treated orally with TCBZ Novartis ®   (10 mg/kg.) and F. hepatica removed from fluke-infested sheep at 3, 24, 48 and 60 h post  TCBZ treatment (PT) were homogenized, and cytosolic fractions were isolated to measure TCBZ metabolites, sulphoxide (TCBZSO) and sulphone (TCBZSO2) concentration by high performance liquid chromatography and carboxylesterase (CE) enzymatic activity by spectrophotometric and SDS-PAGE zymogram procedures (Nousiainen U. and Törrönen R. 1984) We observed a statistically significant increase in enzyme activity at 24 and 48 hours post treatment (P <0.01 and p <0.001, respectively) . This CE increase detected by zymograms is consistent with those values obtained by the spectrophotometric CE assays. The zymogram of cytosolic fractions identified a protein 170 kDa containing the carboxylesterase activity. The 170-kDa. Rf 0.130 CE band, reported in this study for the first time, appears to be inducible by TCBZ exposure when zymograms were analyzed.  The densitograms of the zymograms confirmed the phenomenon of enzyme induction under the experimental conditions of the assay.The results obtained in the present work confirm that Carboxylesterases of F. hepatica is rapidly inducible in the presence of TCBZSO. These results provide not only the understanding of the importance of this metabolic pathway in flukes but carboxylesterase would also be an enzyme that could participate more actively in the development of anthelmintic resistance at TCBZ.