Functional alterations by a subgroup of neonicotinoid pesticides in human dopaminergic neurons

Abstract

Neonicotinoid pesticides, originally developed to target the insect nervous system, have been reported to interact with human receptors and to activate rodent neurons. Therefore, we evaluated in how far these compounds may trigger signaling in human neurons, and thus, affect the human adult or developing nervous system. We used SH-SY5Y neuroblastoma cells as established model of nicotinic acetylcholine receptor (nAChR) signaling. In parallel, we profiled dopaminergic neurons, generated from LUHMES neuronal precursor cells, as novel system to study nAChR activation in human post-mitotic neurons. Changes of the free intracellular Ca2+ concentration ([Ca2+]i) were used as readout, and key findings were confirmed by patch clamp recordings. Nicotine triggered typical neuronal signaling responses that were blocked by antagonists, such as tubocurarine and mecamylamine. Pharmacological approaches suggested a functional expression of α7 and non-α7 nAChRs on LUHMES cells. In this novel test system, the neonicotinoids acetamiprid, imidacloprid, clothianidin and thiacloprid, but not thiamethoxam and dinotefuran, triggered [Ca2+]i signaling at 10–100 µM. Strong synergy of the active neonicotinoids (at low micromolar concentrations) with the α7 nAChR-positive allosteric modulator PNU-120596 was observed in LUHMES and SH-SY5Y cells, and specific antagonists fully inhibited such signaling. To provide a third line of evidence for neonicotinoid signaling via nAChR, we studied cross-desensitization: pretreatment of LUHMES and SH-SY5Y cells with active neonicotinoids (at 1–10 µM) blunted the signaling response of nicotine. The pesticides (at 3–30 µM) also blunted the response to the non-α7 agonist ABT 594 in LUHMES cells. These data show that human neuronal cells are functionally affected by low micromolar concentrations of several neonicotinoids. An effect of such signals on nervous system development is a toxicological concern.

Acute effects of the imidacloprid metabolite desnitro-imidacloprid on human nACh receptors relevant for neuronal signaling

“…Concerning the understanding of the MIE, we used molecular docking studies to provide a rationale for the experimental findings. The availability of several X-ray structures with co-crystallized neonicotinoids has facilitated the establishment of a robust docking model (Ihara et al 2008(Ihara et al , 2014Loser et al 2021a). In the present work, we focused on the overlap of the pesticide N-heteroaromatic ring with the pyridine ring of nicotine.…”

“…As input (oral exposure), we used 0.016 mg DN-IMI/kg body weight. This amount corresponds to 10% of the value used earlier for IMI PBTK modeling (Loser et al 2021a). Our rationale was that DN-IMI can reach about 10% of the IMI content in fruits, vegetables, and cereals (see the introduction for reference).…”

“…The relevance of this finding for human toxicology is further supported by a recent study using cultured human neurons. Clear nAChR signaling and also pronounced receptor desensitization were demonstrated for several neonicotinoids at concentrations that may be reached by dietary or accidental exposure (Loser et al 2021a ).…”

“…The human neuronal precursor cell line LUHMES and the neuroblastoma cell line SH-SY5Y can be differentiated into post-mitotic neurons (Lopes et al 2010 ; Scholz et al 2011 ), and they are often used as a model system to investigate adverse effects on human neurons (Tomizawa and Casida 1999 ; Gustafsson et al 2010 ; Krug et al 2013 , 2014 ; Zhang et al 2014 ; Ring et al 2015 ; Lohren et al 2015 ; Attoff et al 2016 , 2020 ; Smirnova et al 2016 ; Harris et al 2017 ; Tong et al 2017 ; Witt et al 2017 ; Delp et al 2018a , b , 2019 ; Brüll et al 2020 ). The utility of these cell models for functional neurotoxicity testing has been demonstrated for agents that affect voltage-dependent sodium channels or ionotropic receptors (Loser et al 2021a , b ). Both cell types express functional nAChRs and have been used in Ca 2+ -imaging assays to study the effects of several neonicotinoids (Loser et al 2021a ).…”

“…The utility of these cell models for functional neurotoxicity testing has been demonstrated for agents that affect voltage-dependent sodium channels or ionotropic receptors (Loser et al 2021a , b ). Both cell types express functional nAChRs and have been used in Ca 2+ -imaging assays to study the effects of several neonicotinoids (Loser et al 2021a ).…”