Functional neurotoxicity assays to assess substance-induced signaling alterations of human dopaminergic neurons

The potential toxic risk of a substance on the human nervous system is still mainly determined in animal experiments, which in some cases lack predictivity for humans. Approaches based on human neuronal cells have the potential to overcome the limitations of animal models. In manuscript 1, we established an alternative test system based on the human neuronal cell line LUHMES with a high-throughput quantification of free intracellular Ca2+ concentrations ([Ca2+]i) to assess neuronal activity. The suitability of this approach to detect substance-induced activity alterations of ionotropic receptors, voltage-gated ion channels, and electrogenic neurotransmitter transporters was exemplified for three crucial neuronal toxicant targets: Ionotropic purinergic receptors, voltage-gated sodium ion channels, and the electrogenic dopamine transporter (DAT). Besides, the activation of the DAT stimulated synchronized [Ca2+]i oscillations of the neuronal network that were sensitive to different compounds affecting the DAT or voltage-gated ion channels. In manuscript 2, we showed that LUHMES neurons have functional α7 and neuronal non-α7 nicotinic acetylcholine receptors (nAChRs) and verified the applicability of our approach to detect agonistic and antagonistic effects on nAChR signaling. On this basis, we explored the effects of the six most important commercially available neonicotinoids, as some studies indicated effects of these insecticides on mammals, and discovered that four of them trigger [Ca2+]i responses. Thereby, the activation of the α7 nAChR subtype by the four active neonicotinoids was observed. We also detected a desensitizing effect of the active neonicotinoids, as the pretreatment of the cells with these four neonicotinoids negatively modulated a subsequent activation of the nAChRs by nAChR agonists. These data suggest that several neonicotinoids affect LUHMES neurons via the activation of their α7 and non-α7 nAChRs. As neonicotinoids are transformed in the environment and in humans, and some metabolites exhibit a higher toxicity for mammals than insects, we explored in manuscript 3 the effects of the imidacloprid (IMI) metabolite desnitro-imidacloprid (DN-IMI) with our LUHMES-based test system for a broad examination of its effects on human neurons. DN-IMI activated α7 and non-α7 nAChRs present on LUHMES neurons and negatively modulated the responses evoked by nAChR agonists. Our antagonist data verified an activation of nAChRs by DN-IMI with a strong contribution of non-α7 nAChRs. In addition, we studied the agonistic effect of DN-IMI on several human nAChR subtypes heterologously expressed in Xenopus laevis oocytes to clearly identify the affected subtypes. Our results showed the activation of human α7 and several non-α7 nAChRs by DN-IMI with similar potencies compared to nicotine and with significantly higher effectiveness than its parent compound IMI. These findings demonstrate the importance of a comprehensive risk assessment for parent compounds and metabolites. In summary, our LUHMES-based test system allows the evaluation of adverse effects on multiple, important neuronal targets with high reproducibility and concurrence with literature data and results obtained with other methods (patch clamp and oocyte recordings). These findings highlight the value of human neuronal cell-based assays for assessing potential adverse effects on the function of the human nervous system.publishe