Differentiated Neurons Are More Vulnerable to Organophosphate and Carbamate Neurotoxicity than Undifferentiated Neurons Due to the Induction of Redox Stress and Accumulate Oxidatively-Damaged Proteins

Abstract

Organophosphate (OP) and carbamate pesticides are toxic to pests through targeted inhibition of acetylcholinesterase (AChE). However, OPs and carbamates may be harmful to non-target species including humans and could induce developmental neurotoxicity if differentiated or differentiating neurons are particularly vulnerable to neurotoxicant exposures. Hence, this study compared the neurotoxicity of OPs, chlorpyrifos-oxon (CPO), and azamethiphos (AZO) and the carbamate pesticide, aldicarb, to undifferentiated versus differentiated SH-SY5Y neuroblastoma cells. OP and carbamate concentration-response curves for cell viability were undertaken using 3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) and lactate dehydrogenase (LDH) assays and cellular bioenergetic capacity assessed via quantitation of cellular ATP levels. Concentration-response curves for inhibition of cellular AChE activity were also generated and the production of reactive oxygen species (ROSs) was monitored using a 2′,7′-dichlorofluorescein diacetate (DCFDA) assay. The OPs and aldicarb reduced cell viability, cellular ATP levels, and neurite outgrowth in a concentration-dependent fashion, from a threshold concentration of ≥10 µM. Neurotoxic potency was in the order AZO > CPO > aldicarb for undifferentiated cells but CPO > AZO > aldicarb for differentiated cells and this toxic potency of CPO reflected its more extensive induction of reactive oxygen species (ROSs) and generation of carbonylated proteins that were characterized by western blotting. Hence, the relative neurotoxicity of the OPs and aldicarb in part reflects non-cholinergic mechanisms that are likely to contribute to developmental neurotoxicity.

Differential Effects of Paraquat, Rotenone, and MPTP on Cellular Bioenergetics of Undifferentiated and Differentiated Human Neuroblastoma Cells

“…Notably, DA cells, followed by the CH phenotype, were the most vulnerable to neurotoxicant damage and loss of cell viability. All of the neurotoxic agents significantly decreased ATP levels, and the decline in ATP levels was similar to that induced by MTT and thereby provided an alternative means to validate the neurotoxicant-induced decline in cell viability, as observed in other studies [24,55]. The neurotoxicants inhibited either mitochondrial complex I or complex III activity or both, with a parallel increase in cellular lactate production.…”

“…The process of differentiation results in a change in the proteome with the expression of a number of mature neuronal markers, including cytoskeletal proteins such as βIII-tubulin and microtubule-associated proteins (MAP-2 and MAP-tau) required for the production of neuritic projections. Some of these proteins may be oxidatively damaged in response to neurotoxicants and contribute to their acute or chronic toxicity [24]. Hence, at present, we can only speculate on the rationale for the differential toxicity of PQ, RO, and MPTP and the relative vulnerability of DA cells, but this could arise from the altered expression of proteins vulnerable to redox damage.…”

“…However, data from bioenergetics and oxidative stress analyses confirmed that the DA cells were the most susceptible to mitochondrial disruption, induction of oxidative damage, and therefore cytotoxicity. Our previous studies have reported that there are brain regions more vulnerable to the cytotoxic effects of certain pesticides [63] and that at a cellular level, differentiation renders SH-SY5Y cells more vulnerable to neurotoxicity from organophosphate and carbamate pesticides [24]. The process of differentiation results in a change in the proteome with the expression of a number of mature neuronal markers, including cytoskeletal proteins such as βIII-tubulin and microtubule-associated proteins (MAP-2 and MAP-tau) required for the production of neuritic projections.…”

“…On day seven, the media were removed, and the cells were trypsinized, split at a 1:1 ratio, and incubated in fresh differentiation media. On day eight, the media were aspirated, and the cells were treated with 10 µM RA in low serum SH-SY5Y medium (1% FBS) for 48 h. On day ten, cells were trypsinized, split at a 1:1 ratio, and incubated in the same differentiation media for 24 h. The media were aspirated, and the cells were treated with medium containing 50 ng/mL brain-derived neurotrophic factor (BDNF) (B3795, Sigma-Aldrich, Poole, UK) and low serum media containing RA for a further 2 days, after which the cells displayed a fully differentiated morphology [23] as described in a previous publication [24] (Supplementary Figure S1). Plated cells were protected from light throughout the differentiation period to prevent the disassociation of RA.…”

An In Silico and In Vitro Assessment of the Neurotoxicity of Mefloquine

“…A reduction in cell metabolic activity, as measured by the MTT assay, is often used as a surrogate for changes in cell viability [ 32 ]. However, to provide additional confirmation of the impact of MQ on SH-SY5Y cell viability, an LDH assay was employed.…”