Study on Glutathione S-Transferase (GST) Inhibition Assay by Triclabendazole. І: Protoscoleces (Hydatid Cyst; Echinococcus granulosus) and Sheep Liver Tissue

Abstract

Hydatid disease is a term used to refer infection with the methacestode of Echinococcus granulosus parasite in humans, and echinococcusis is restricted to infection with the adult stage in carnivores.Glutathione S-Transferase (GST) represents the major class of detoxification enzymes from helminth parasites such as Echinococcus protoscoleces (PSC) and it is candidate for chemotherapeutic and vaccine design. Therefore, GST of protoscoleces could be a target for evaluation of drug effect as triclabendazole in hydatid cyst. For this purpose, GST enzymes were purified from protoscoleces of hydatid cyst and sheep liver tissue by glutathione affinity chromatography using a wash-batch method and subsequently detected their SDS-PAGE pattern. Afterward, GST specific activity levels were assayed in the whole extract and purified solutions spectrophotometrically at 30°C with reduced glutathione (GSH) and 1-chloro-2, 4-dinitrobenzen (CDNB) substrate. Finally, GST inhibition assay was investigated in the solutions by powder and bolus of triclabendazole. GST fraction as a 26 kDa (MW) band was obtained on SDS-PAGE. The level of GST specific activity in purified solutions was detected 10.24 µmol/min/mg proteins for protoscoleces and 37.84 µmol/min/mg protein for liver tissue. Comparison of the effect of powder and bolus of triclabendazole in solutions revealed inhibition concentration (IC50) 8.71 and 11.16 µg/ml for protoscoleces GST and 8.65 and 9.70 µg/ml for liver tissue GSTs, respectively. These findings suggest the possibility of selective inhibition of protoscoleces. GSTs by triclabendazol in vitro and use of these results for understanding of its molecular effect in vivo.