Abstract:
Micropollutants in the aquatic environment pose a risk to human and environmental health. Effect-based tools have been applied in environmental monitoring for diverse toxicity endpoints but testing method for neurotoxicity is still limited. The goal of this PhD thesis was to develop and implement high-throughput methods for testing neurotoxicity of typical environmental organic pollutants and mixtures of chemicals extracted from water samples. Neurite outgrowth inhibition and acetylcholinesterase (AChE) inhibition were considered as key neurotoxicity endpoints and human neuroblastoma SH-SY5Y cells were used for both assays. The assays were set up in 384-well plates for high-throughput and repeatable concentration-response assessment. The AChE inhibition assay using purified enzyme has been applied widely, but there has been an issue that natural organic matters such as dissolved organic carbon (DOC) contained in environmental samples can suppress AChE inhibition in the assay. In the cellular assay, AChE inhibition by paraoxon-ethyl was not impacted by DOC up to 68 mgc/L and binary mixtures of paraoxon-ethyl and water extracts showed concentration-additive effects, which indicates no disturbance by DOC and applicability of the cell-based AChE inhibition assay for testing of environmental samples. Chemicals with potential developmental neurotoxicity (DNT) are often hydrophobic. Hydrophobic chemicals can easily intercalate into the cell membrane and provoke effects via nonspecific manner, i.e., baseline toxicity. To investigate whether DNT of chemicals is driven via specific modes of action or merely via baseline toxicity, test chemicals were selected based on their potential DNT from literature or a combination of occurrence data and effects detected in water samples. The effects on neurite outgrowth and cytotoxicity were directly measured in SH-SY5Y cells and the observed effects were compared with predicted baseline cytotoxicity. Since existing prediction models for baseline toxicity had limited application, a prediction model was newly established using a mass balance model based on constant critical membrane concentrations, which can be applied for chemicals of a wide range of hydrophobicity and speciation. When comparing the measured effects in SH-SY5Y with the predicted baseline toxicity, more hydrophobic chemicals tended to trigger toxicity on neurite outgrowth and cell viability via baseline toxicity. The hydrophobic chemicals were still often highly potent while some more hydrophilic chemicals exhibited high specificity but often lower potency. Environmental pollutants with specific modes of action targeting neurite outgrowth were identified by comparing the effects on neurite outgrowth and cytotoxicity. Highly specific effects were observed for two carbamate insecticides, the pharmaceutical mebendazole, the biocide 1,2-benzisothiazolin-3-one, and many other chemicals that were detected in surface water and wastewater treatment plant (WWTP) effluent samples. The two types of water samples were tested in neurite outgrowth assay and the effects on neurite outgrowth were even observed when the samples were diluted by a factor of 5. While overall cytotoxicity was similar between two types of samples, higher toxicity on neurite outgrowth was observed for surface water than WWTP effluent. This led to more specific inhibition of neurite outgrowth by surface water, indicating that higher concentrations of chemicals and/or more potent chemicals acting on neurite outgrowth were contained in the surface water samples. Subsequently, the measured mixture effects were further explained by measured effects of single chemicals and overall, chemicals with high effect potency and/or high occurrence were identified as major mixture effect drivers. While main contributors were different between individual samples for surface water, mebendazole was a dominant contributor for the effects observed in WWTP effluent. The detected chemicals still explained only a small fraction of the measured mixture effects of surface water (up to 4.4%) and WWTP effluent (up to 6.8%). When the two neurotoxicity endpoints were assessed in identical samples, the effects appeared not to be related to each other and both neurotoxicity endpoints were sensitive enough to capture toxicity even when the samples were diluted. The experiments with single chemicals and the applications in case studies demonstrated that both neurotoxicity assays are suitable for environmental monitoring of neurotoxicants. Further testing of various chemicals and environmental mixtures can be useful to identify more effect drivers in the environment. Consideration of more diverse neurotoxicity endpoints would enable more comprehensive assessment of water quality. In the future, these assays have also the potential to be used for human biomonitoring and can be applied to other complex environmental matrices such sediments or biota.
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