Exposure matters: The impact of exposure characteristics on In Vitro neurotoxicity of pesticides and implications for regulatory testing.

Current risk assessment does not capture neurodegenerative effects of pesticides. Since pesticide exposure is linked to Parkinson's Disease (PD), test methods for regulatory assessment are needed. Acute toxicity tests fail to reflect the diversity in real-life exposure. Therefore, our aim was to determine to what extent duration, time after, and frequency of exposure affect pesticide-induced effects related to neurodegeneration in vitro. To this end, differentiated SH-SY5Y human neuroblastoma cells were exposed to pesticides (dinoseb, endosulfan, mancozeb, rotenone; 0.1 μM - 100 μM). Cells were exposed for (a) 24 h, (b) 24 h followed by 18 h without exposure, (c) 24 h followed by 72 h without exposure, (d) four six-hour exposures with 18 h in exposure-free medium, (e) 96 h. Mitochondrial metabolic activity and cytotoxicity were determined by alamar blue and lactate dehydrogenase assays. Pesticide concentrations were determined by mass spectrometry methods. Our results show that increasing exposure duration increased the potency, but the degree differed per pesticide. Increasing time after exposure increased the potency, but only for mancozeb and rotenone. Increasing exposure frequency (while maintaining the total exposure time) caused a left-shift only for mancozeb. These left-shifts could not be explained by an increase in the amount of pesticide measured in the cells. These data indicate that variations in exposure scenarios impact potencies of pesticides in vitro. To ensure that in vitro strategies for risk assessment generate relevant data, these strategies should be informed by real-life exposure. To define a relevant range of exposure scenarios, more extensive evaluation is required.