Imidacloprid, thiacloprid, and their imine derivatives up-regulate the α4β2 nicotinic acetylcholine receptor in M10 cells

Neonicotinoids are the most important new class of insecticides of the last decade. They act as nicotinic acetylcholine receptor (AChR) agonists. This investigation tests the hypothesis far the first time that neonicotinoid insecticides and their imine derivatives up-regulate the (alpha4 beta2 nicotinic AChR subtype, which represents >90% of the high-affinity [H-3]nicatine binding sites in mammalian brain. The alpha4 beta2 receptor stably expressed in mouse fibroblast M10 cells was assayed after 3 days' exposure to the test compound, as [H-3]nicotine binding following immunoisolation by monoclonal antibody (mAb 299) or as [(125)]mAb 299 labeling for cell surface receptors. We found that imidacloprid (IMI) tone of the most important insecticides) and thiacloprid (THIA) increased [H-3]nicotine binding levels (up-regulation of the alpha4 beta2 AChRs) by five- to eightfold with EC50s of similar to 70,000 and 19,000 nM, respectively, compared with 760 nM for (-)-nicotine. In contrast, two imine analogs [the desnitro metabolite of IMI (DMIMI) and the descyano derivative of THIA] gave up-regulation by eightfold and EC50s of 870 and 500 nM, respectively. The potency order for up-regulation by the five aforementioned compounds was correlated with their in vitro IC50s for inhibiting [H-3]nicotine binding (r(2) = 0.99, n = 5), indicating that binding to the alpha4 beta2 receptor initiates the up-regulation. A potent olefin derivative of the THIA imine up-regulated with an EC50 of 22 nM. DNIMI-induced upregulation mainly occurred intracellularly rather than at the cell surface. These findings in alpha4 beta2-expressing M10 cells indicate the possibility that some neonicotinoid insecticides or their metabolites, on accidental human exposure or when used for flea control on dogs, may also up-regulate the receptor in mammals. (C) 2000 Academic Press.