ABSTRACT
Aim: The cholinesterase (ChE) based inhibition and histopathological studies from fish were investigated and represented in this study to develop as one of the great potential biomarkers for heavy metals monitoring. Methodology: In this study, the histopathological study of gills were observed a under microscope. The capability of ChE extracted from the gills of Clarias gariepinus was assessed for declining Cd. ChE was purified through affinity chromatography and continued with the optimisation and inhibition study (IC50) of cholinesterase. Results: Histopathological study of gills was carried out and several changes such as aneurysm, necrosis and lamella fusion were noted. Purification fold obtained from purified enzyme was 1.15 with 30% a yield specific activity 20.726. The optimum temperature for purified AChE was 35°C along with acetylthiocholine iodide (ATC) as a preferable substrate that had the highest Vmax value of 0.5452 U mg-1 and the lowest Km value of 0.0311 mM. The optimum pH was observed to be 10 of Tris-HCl as a medium. Meanwhile, the IC50 of cadmium was 6.808 mg l-1 with R2 value of 0.9532. Interpretation: The result of the study can be used as a tool for further developing a biomarker for the detection of heavy metals in aquatic ecosystems. In addition, the baseline data provided can also be used for designing a kit, which would give rapid and accurate result.
ABSTRACT
Aim: Cholinesterase (ChE) inhibition and histopathological features of brain tissues of Clarias gariepinus were investigated with a purpose to develop biomarkers for monitoring heavy metals. Methodology: The effects of cadmium toxicity on C. gariepinus were assessed by ChE inhibition and characterisation of ChE by affinity chromatography and histological variations in brain tissues were studied by H&E staining under light microscope. Results: The purified enzyme gave 10.58 fold purification with the recovery of 12% with specific activity of 159.729 U mg-1. The Michaelis-Menten constant (Km) and Vmax value obtained was 1.985 mM and 0.4479 µmol min-1 mg-1, respectively. The enzyme showed the ability to hydrolyse acetylthiocholine iodide (ATC) at a faster rate compared to other two synthetic substrates, propionylcholine iodide (PTC) and butyrylthiocholine iodide (BTC). ChE showed highest activity at 35°C in Tris-HCl pH 7.5. The metal inhibition study also found the maximal inhibitory concentration (IC50) value for cadmium to be 0.6517 mg l-1. Histological study revealed decrease in the number of cells, degeneration of Purkinje cells, increased aggregation area and cells necrosis. Interpretation: Inhibition of cholinesterase activity and degeneration of Purkinje cells observed in the present study can be used as a tool for further developing a biomarker for detecting heavy metals in the aquatic ecosystems.