Evaluation of methods for the assessment of in vitro neurotoxicity: Calcium homeostasis as target for insecticides

Abstract

Due to the REACH regulation, more animal studies for regulatory safety studies are needed in the coming years unless suitable alternatives become available. In addition, regulatory neurotoxicity tests have been criticized for their low sensitivity and the large amount of animals, time and money that are needed to perform these studies. There is thus a need for sensitive regulatory neurotoxicity tests that are fast, cheap and require (no or) less animals. Therefore, in this thesis several in vitro targets and methods for the development of in vitro screening tools for (developmental) neurotoxicity have been investigated as part of the EU project DENAMIC. In vitro methods for the investigation of chemical-induced oxidative stress, neurotransmitter release and calcium homeostasis were evaluated. Considering their relevance for proper neuronal function, these endpoints are very suitable for chemical neurotoxicity screening. Before these endpoints can be used for neurotoxicity testing in vitro, methods for the detection of chemical-induced effects on these endpoints should be carefully evaluated. Several methods for the detection of oxidative stress and neurotransmitter release were evaluated in this thesis. However, no reliable, suitable, real-time and high-throughput methods were found for the detection of oxidative stress and neurotransmitter release. Generally, real-time kinetic changes in calcium homeostasis are measured by either a plate reader-based method or a fluorescence microscopy-based method. However, plate reader-based methods have been criticized because of their low(er) temporal and spatial resolution, low(er) sensitivity and potential pitfalls and artifacts. In this thesis, a direct comparison was made between results obtained in a plate reader- and a fluorescence microscopy-based method. Results obtained with the plate reader-based method did not show consistent changes in basal [Ca2+]i or in the depolarization-evoked increase in [Ca2+]i for the test compounds. Therefore, all other studies in this thesis on calcium homeostasis were performed with a fluorescence microscopy-based method. The effects of insecticides on calcium homeostasis were extensively investigated. It was demonstrated that different types of insecticides (organophosphates, organochlorines, pyrethroids) can acutely change basal [Ca2+]i and/or acutely inhibit VGCCs (depolarization-evoked increase in [Ca2+]i) at low concentrations. Furthermore, it was shown that an acute exposure is often sufficient for screening chemicals for neurotoxicity and that for chemicals with common modes of action, additivity could be expected in binary mixtures. Long-term potentiation (LTP), a measure of learning and memory, was studied ex vivo to understand the relationship and bridge the gap between in vitro and in vivo studies. No correlation between LTP, animal behavior and effects on calcium homeostasis were found. Altogether, this thesis demonstrates that calcium homeostasis is a target for neurotoxic insecticides and could be used for neurotoxicity screening studies. In addition, the data indicates that the effects of mixtures should be included in risk assessment to account for additive effects. It was proposed and recommendations are given to develop a battery of in vitro tests that could be used to identify and prioritize chemicals for neurotoxicity studies.