Wijesekara Mudiyanselage, Anusha Udayangani Kumari Manike Wijesekara
(2021)
An evaluation of the neurotoxicity of pesticides and alcohol using human neuroblastoma cells and human progenitor cortical neural stem cells.
PhD thesis, University of Nottingham.
Abstract
Health concerns have been raised that the developing brain may be particularly vulnerable to the toxic effects of organophosphate (OP) and carbamate pesticides and alcohol. Invariably, many of the adult population are co-exposed to both pesticides and alcohol over the course of a lifetime, and for some individuals, also exposed in an acute setting. Chlorpyrifos and Azamethiphos are commonly used OP pesticides and Aldicarb is a commonly used carbamate pesticide. Safety testing of these commonly encountered neurotoxicants is a challenge to the area of development neurotoxicity. Induction of reactive oxygen species (ROS) has been proposed as a mechanism of acute OP, carbamate and alcohol induced poisoning cases, but there is uncertainty related to the cytotoxic responses associated with oxidative stress induced by these neurotoxicants. Herein, SH-SY5Y neuroblastoma cells and human embryonic progenitor cortical neuronal stem cells (hNPC) were used for evaluating ROS-mediated oxidative stress and cellular damage including elevation of protein carbonylation.
Both undifferentiated and differentiated cells of neuroblastoma SH-SY5Y and hNPCs were challenged with active (oxon) metabolites of chlorpyrifos (CPO), azamethiphos (AZO) and Aldicarb from (1 - 200 µM) for 24 hours, and ethanol for 10 - 200 mM for 3, 6, 12 and 24 hours to mimic episodic binge drinking. Undifferentiated and differentiated neuroblastoma SH-SY5Y cells were also co-exposed to pesticides such as concentrations producing 5,10, 20, or 50% cell death (IC5 IC10, IC20 and IC50) of CPO, AZO and Aldicarb in combination with 10 - 200 mM ethanol for 24 hours. The effect of pesticides and ethanol on cell metabolic activity and viability was determined by (4,5 dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT), lactate dehydrogenase (LDH), and ATP assays. Cellular morphological changes that arose from the exposures were recorded by phase contrast microscopy. The effect of pesticides and ethanol on cellular differentiation was assessed by neurite outgrowth inhibition. The effect of the three pesticides on acetylcholinesterase inhibition was determined by Elman’s assay. The generation of ROS was quantified using a 2’,7’-dichlorofluorescein diacetate (DCFDA) assay. Cellular protein modification and damage was determined using spectrophotometric protein carbonyl content (PCC) assay. Specific targets of oxidatively damaged proteins were identified using gel electrophoresis, and oxyblots.
CPO, AZO and Aldicarb was cytotoxic to both cell lines in a concentration-dependent fashion. Alcohol induced concentration and time dependent increases in cytotoxic responses in both cell lines. Cellular energy supply (ATP levels) decreased in a concentration-dependent manner in cells exposed to the three tested pesticides and in a concentration and time dependent manner for ethanol treatments. CPO, AZO and Aldicarb induced AChE inhibition in both SH-SY5Y and hNPCs cells. ROS levels increased, with an associated increase of protein carbonylation, particularly within cytosolic proteins of 35 - 120 kDa. All pesticides exposures including those at low levels (IC10) induced oxidation of an ~50 kDa protein, while low concentrations of ethanol (such as 10 mM) also induced protein carbonylation in hNPCs. Co-exposure of low doses of pesticides and ethanol induced cell metabolic and cell viability loss in neuroblastoma SH-SY5Y cells with increasing pesticide and ethanol concentration and induced a mix of synergistic or antagonistic effects. Co-exposure of pesticides and ethanol induced oxidative damage to both undifferentiated and differentiated cells as evidenced by DCFDA assay, PCC and oxyblots.
This study provides evidence that ROS generation correlates with induction of cytotoxicity in neuronal cells after exposures to CPO, AZO, Aldicarb and ethanol. Furthermore, specific protein targets were subjected to oxidative damage after acute pesticide and alcohol exposures to neuronal cells. The cytotoxic responses are dependent not only on the concentration and duration, but also on the developmental stage. hNPC and SH-SY5Y neuroblastoma cells provided in-vitro model systems for the better understanding of cellular and molecular mechanisms involved in development neurotoxicity studies and can be regarded as complementary to animal testing in a cost and time effective manner.
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