Interaction of imidacloprid metabolites and analogs with the nicotinic acetylcholine receptor of mouse brain in relation to toxicity

The favorable selective toxicity of imidacloprid (IMI) to insects versus mammals is attributed to differences in their binding affinity or potency in the nicotinic acetylcholine receptor (nAChR), a proposal tested here by studies on the mechanism of toxicity of IMI metabolites and analogs to mammals. IMI, its desnitro metabolite (DN-IMI), its nitromethylene analog (CH-IMI), and 26 other analogs and metabolites were examined for intraperitoneal toxicity to mice and potency for in vitro inhibition of the binding of [3H]nicotine (the classical nAChR probe) in mouse brain membranes. IMI and 7 analogs with LD50 values of 7-50 mg/kg (or intoxication signs at 50 mg/kg) inhibited [3H]nicotine binding by 50% (IC50) at 12-800 nM whereas 21 other analogs that were not toxic at 50 mg/kg gave an IC50 of >1000 nM, thereby correlating the toxicity with interaction at the [3H]nicotine binding site. The most potent compounds were DN-IMI and CH-IMI (and its tetrahydropyrimidine analog) with LD50s of 7-24 mg/kg and IC50s of 12-33 nM compared with values for IMI of 39-49 mg/kg and 806 nM, respectively. DN-IMI is therefore a candidate bioactivation product for IMI in mammals. Scatchard analyses indicated that CH-IMI in vitro and possibly DN-IMI in vitro and ex vivo compete for the nicotine site (which is at or near the ACh site). When used directly as radioligands, single, saturable, high-affinity binding sites were observed for [3H]DN-IMI (KD 13 nM, Bmax 51 fmol/mg protein) and [3H]CH-[IMI (KD 16 nM, Bmax 20 fmol/mg protein) using the conditions of [3H]nicotine binding (KD 7.8 nM, Bmax 87 fmol/mg protein). [3H]DN-IMI also binds to kidney membranes at a site where it is displaced by atropine (ki 0.5 micromolar). [3H]CH-IMI is particularly useful for comparative studies because of high-affinity sites in both insect and mammalian brain.