Neonicotinoid metabolic activation and inactivation established with coupled nicotinic receptor-CYP3A4 and -aldehyde oxidase systems

Two important enzymes in metabolism of the principal neonicotinoid insecticide imidacloprid are liver microsomal CYP3A4 and cytosolic aldehyde oxidase (AOX). CYP3A4 oxidation at several molecular sites and AOX reduction at the nitro substituent result in either an increase (activation) or decrease (inactivation) of agonist potency at nicotinic acetylcholine receptors (nAChRs), both insect and vertebrate α4β2. This study evaluates activation or inactivation of 11 neonicotinoids in a continuous two-step system coupling metabolism and receptor binding. For metabolism, the neonicotinoid is incubated with CYP3A4 and NADPH or AOX with the cosubstrate N-methyl-nicotinamide, terminating the reaction with ketoconazole or menadione, respectively, to inhibit further conversion. For receptor assay, either the Drosophila nAChR and [3H]imidacloprid or the α4β2 nicotinic receptor and [3H](−)-nicotine are added to determine changes in neonicotinoid potency. With the Drosophila nAChR assay, the N-methyl compounds N-methyl-imidacloprid and thiamethoxam are activated 4.5–29-fold by CYP3A4 whereas nine other neonicotinoids are not changed in potency. With the vertebrate α4β2 nAChR, AOX enhances imidacloprid potency but CYP3A4 does not. The AOX system coupled with the Drosophila receptor strongly inactivates clothianidin, dinotefuran, imidacloprid, desmethyl-thiamethoxam, and thiamethoxam with some inactivation of nitenpyram and nithiazine, and little or no effect on four other compounds.