Lizard cholinesterases as biomarkers of pesticide exposure: enzymological characterization

Here we report the results of a study conducted to elucidate the enzymological characteristics of lizard cholinesterases (ChEs) in order to use them as potential biomarkers for pesticide exposure. Serum and brain tissue of the lizard Gallotia galloti were used as ChE sources and in vitro assays were performed to identify acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) activities. The pH, substrate concentration, and specificity for ChE assays as well as the response of serum BChE to the reactivating agent pyridine-2-aldoxime methochloride (2-PAM) were also investigated in order to assess the possibilities of this methodology in biomonitoring programs. By the use of selective substrates and the inhibitor tetraisopropyl pyrophosphoramide, AChE and BChE activities were identified in lizard serum, while brain contained solely AChE. Likewise, butyrylthiocholine iodide was the optimum substrate for determining BChE activity and acetylthiocholine iodide for assaying both serum and brain AChE activities. The optimal ranges of pH and substrate concentrations were 7.5 to 8.0 and 5 to 10 mM, respectively. Serum was incubated with different doses of the organophosphorus (OP) compounds dichlorvos and paraoxon and subsequently incubated in the presence of two concentrations of 2-PAM (2 x 10(-2) or 2 x 10(-4) M). Reactivation rate of phosphorylated BChE was related to the degree of inhibition of BChE and the dose of 2-PAM. It was found that a 90-min incubation time with 2 x 10(-4) M of 2-PAM satisfactorily increased the OP-inhibited BChE activity. The enzymological properties of serum BChE activity and its in vitro reactivation in the presence of 2-PAM represent the initial justification for its use in monitoring OP contamination in the field.