Abstract
Despite the significant progress in both scientific understanding and regulations, the safety of agricultural pesticides continues to be called into question. The need for complementary analytics to identify dysregulation events associated with chemical exposure and leverage this information to predict biological responses remains. Here, we present a platform that combines a model organ-on-chip neurovascular unit (NVU) with targeted mass spectrometry (MS) and electrochemical analysis to assess the impact of organophosphate (OP) exposure on blood-brain barrier (BBB) function. Using the NVU to simulate exposure, an escalating dose of the organophosphate chlorpyrifos (CPF) was administered. With up to 10 μM, neither CPF nor its metabolites were detected across the BBB (limit of quantitation 0.1 µM). At 30 µM CPF and above, targeted MS detected the main urinary metabolite, trichloropyridinol (TCP), across the BBB (0.025 µM) and no other metabolites. In the vascular chamber where CPF was directly applied, two primary metabolites of CPF, TCP and diethylthiophosphate (DETP), were both detected (0.1–5.7 µM). In a second experiment, a constant dose of 10 µM CPF was administered to the NVU, and though neither CPF nor its metabolites were detected across the BBB after 24 h, electrochemical analysis detected increases in acetylcholine levels on both sides of the BBB (up to 24.8 ± 3.4 µM) and these levels remained high over the course of treatment. In the vascular chamber where CPF was directly applied, only TCP was detected (ranging from 0.06 μM at 2 h to 0.19 μM at 24 h). These results provide chemical evidence of the substantial disruption induced by this widely used commercial pesticide. This work reinforces previously observed OP metabolism and mechanisms of impact, validates the use of the NVU for OP toxicology testing, and provides a model platform for analyzing these organotypic systems.
Highlights
Organophosphates (OPs) are a class of compounds commonly used in commercial pesticides and include nerve gas chemical warfare agents such as sarin, VX, and Novichok agents
Eluate from the vascular and neuronal sides was assessed using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) for targeted toxicant profiling and electrochemical analysis for targeted metabolite profiling. These data validate the predictive power of the neurovascular unit (NVU), the high analytical utility of combined MS, and electrochemical measurements and provide insight into the substantial disruption induced by this widely used commercial pesticide
3) What effects does CPF exposure have on cellular metabolism at and across the blood-brain barrier (BBB)? To this end, environmental exposure to CPF was simulated within the NVU and morphological and metabolic analysis was performed
Summary
Organophosphates (OPs) are a class of compounds commonly used in commercial pesticides (e.g., parathion, chlorpyrifos, and diazinon) and include nerve gas chemical warfare agents such as sarin, VX, and Novichok agents. To gain additional insights into CPF-induced chemical and morphological perturbations, in vitro organotypic models offer medium-throughput systems that complement traditional cell culture techniques and may replace or reduce animal testing (Pridgeon et al, 2018; Low et al, 2021). CPF has been shown to cause alterations to the integrity of the BBB upon exposure, enabling CPF and other toxicants to enter the brain (Levin et al, 2001; Li and Ehrich, 2013) These risks associated with CPF exposure combined with its continued use in the United States demand further investigation and refinement of our ability to identify dysregulation events associated with chemical exposure and leverage this information to predict biological responses (Rauh et al, 2012; Smith et al, 2014)
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