In Vitro Neurotoxicity of Flumethrin Pyrethroid on SH-SY5Y Neuroblastoma Cells: Apoptosis Associated with Oxidative Stress

Abstract

Pyrethroids are neurotoxicants for animals, showing a pattern of toxic action on the nervous system. Flumethrin, a synthetic pyrethroid, is used against ectoparasites in domestic animals, plants, and for public health. This compound has been shown to be highly toxic to bees, while its effects on other animals have been less investigated. However, in vitro studies to evaluate cytotoxicity are scarce, and the mechanisms associated with this effect at the molecular level are still unknown. This study aimed to investigate the oxidative stress and cell death induction in SH-SY5Y neuroblastoma cells in response to flumethrin exposure (1-1000 µM). Flumethrin induced a significant cytotoxic effect, as evaluated by MTT and LDH leakage assays, and produced an increase in the biomarkers of oxidative stress as reactive oxygen species and nitric oxide (ROS and NO) generation, malondialdehyde (MDA) concentration, and caspase-3 activity. In addition, flumethrin significantly increased apoptosis-related gene expressions (Bax, Casp-3, BNIP3, APAF1, and AKT1) and oxidative stress and antioxidative (NFκB and SOD2) mediators. The results demonstrated, by biochemical and gene expression assays, that flumethrin induces oxidative stress and apoptosis, which could cause DNA damage. Detailed knowledge obtained about these molecular changes could provide the basis for elucidating the molecular mechanisms of flumethrin-induced neurotoxicity.

Keywords: SH-SY5Y cells; apoptosis; flumethrin; neurotoxicity; oxidative stress.

Figure 1

The cytotoxicity induced by flumethrin (1–1000 µM) on the viability of the SH-SY5Y cell line after a 24 h incubation period. Cell viability was determined by MTT reduction, and the MTT reduction (%) dose-response curve was used to obtain the IC₅₀ value (A), or as LDH release (B). Data was normalized as % control (c). DMEM-treated cells with 1% FBS were the positive control (c), and cells with 0.1% DMSO were the negative control (Veh). Results are presented as the mean ± SD of six replicates. * p < 0.05, ** p < 0.01, *** p < 0.001 compared to Veh.

Figure 2

The ROS production induced by flumethrin (1–1000 µM) in SH-SY5Y cells after a 24 h incubation period. ROS production is expressed as % of control. DMEM-treated cells with 1% FBS were the positive control (c), and cells with 0.1% DMSO were the negative control (Veh). Results are presented as the mean ± SD of six replicates. *** p < 0.001 compared to Veh.

Figure 3

The NO production induced by flumethrin (1–1000 µM) in SH-SY5Y cells after a 24 h incubation period. Data was normalized as % control. DMEM-treated cells with 1% FBS were the positive control (c), and cells with 0.1% DMSO were the negative control (Veh). Results are presented as the mean ± SD of six replicates. * p < 0.05, *** p < 0.001 compared to Veh.

Figure 4

The MDA production induced by flumethrin (1–1000 µM) in SH-SY5Y cells after a 24 h incubation period. The content of MDA (µM) was calculated for each sample from a standard curve. DMEM-treated cells with 1% FBS were the positive control (c), and cells with 0.1% DMSO were the negative control (Veh). Results are presented as the mean ± SD of six replicates. * p < 0.05, *** p < 0.001 compared to Veh.

Figure 5

The caspase 3/7 activity induced by flumethrin (1–1000 µM) in SH-SY5Y cells after a 24 h incubation period. DMEM-treated cells with DMEM with 1% FBS were the positive control (c), and cells with 0.1% DMSO were the negative control (Veh). Results are presented as the mean ± SD of six replicates. * p < 0.05, *** p < 0.001 compared to Veh.

Figure 6

The effect of flumethrin (20, 50, and 500 µM) in SH-SY5Y cells after a 24 h incubation period on Bax (A), Bcl-2 (B), Casp-3 (C), BNIP3 (D), APAF1 (E), and AKT1 (F) gene expressions related to apoptosis. All data were normalized with GAPDH expression and presented as relative to control. Data are expressed as fold-change with respect to the control (c). Cells with 0.1% DMSO were the negative control (Veh). Results are presented as the mean ± SD of four replicates. * p < 0.05, ** p < 0.01 *** p < 0.001 compared to vehicle.

Figure 7

The effect of flumethrin (20, 50, and 500 µM) in SH-SY5Y cells after a 24 h incubation period on NFκB (A), NRF2 (B), and SOD2 (C) gene expressions related to oxidative stress and the antioxidant gene transcription. All data were normalized with GAPDH expression and given as relative to control. Data are expressed as fold-change with respect to the control (c). Cells with 0.1% DMSO were the negative control (Veh). Results are presented as the mean ± SD of four replicates. * p < 0.05, *** p < 0.001 compared to vehicle.