Optimization of acetylcholinesterase and metabolic enzyme activity in multiple fish species

Abstract

An examination of acetylcholinesterase (AChE), citrate synthase (CS) and lactate dehydrogenase (LDH) activities in the brains of a range of fish species, occupying different trophic levels, is useful to investigate the full extent of the effects of pulse exposures to pesticides. This investigation explores the optimization of microplate procedures for AChE, CS and LDH measurements in the brain tissue of species commonly used in laboratory investigations and those common to Canadian watersheds. A microplate assay was optimized for the measurement of AChE in the brain homogenates of seven fish species. The critical aspects of this assay requiring optimization were pH, substrate concentration and tissue dilution. Incubation with specific cholinesterase inhibitors indicated that enzymatic activity in the brain homogenates of each species was attributed to AChE only. Mcroplate assays were also optimized for the measurement of the metabolic enzymes, CS and LDH, in the brain homogenates of six fish species. For these assays, low interspecies variability was exhibited between optimized factors including pH, substrate, chromogen and cofactor concentrations. For each assay optimized, enzyme activities in the brain homogenates were stable for 2 to 3 hours post-thaw. Results from the present study will aid future ecotoxicological investigations of the potential impacts of AChE inhibition on neuronal glucose metabolism. Copyright © 2007, CAWQ.