Cytotoxicity induced by three commercial neonicotinoid insecticide formulations in differentiated human neuroblastoma SH-SY5Y cells

Abstract

Background: Neonicotinoid insecticides are used worldwide for crop protection. They act as agonists at postsynaptic nicotinic acetylcholine receptors (nAChRs), disrupting normal neurotransmission in target insects. Human exposure is high due to the widespread use of neonicotinoids and their residues in food. This study aimed to evaluate the in vitro neurotoxicity of three neonicotinoid commercial formulations Much 600 FS® (imidacloprid 600 g L^-1), Evidence 700 WG® (imidacloprid 700 g kg^-1), and Actara 250 WG® (thiamethoxam 250 g kg^-1) in differentiated human neuroblastoma SH-SY5Y cell line.

Methods: Cells were incubated with the pesticides for 96 h, and the cytotoxicity was evaluated through the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium·bromide (MTT) reduction and neutral red (NR) uptake assays. Toxicological pathways such as reactive oxygen (ROS) and nitrogen species (RNS) production, mitochondrial membrane potential, cell death mode, and the expression of the pro-apoptotic protein Bax were also evaluated.

Results: EC₅₀ values of 266.4, 4,175, and 653.2 mg L^-1 were found for Much®, Evidence® and Actara®, respectively. Significant increases in ROS and RNS generation were observed for all pesticides, while mitochondrial membrane potential and Bax protein expression showed no significant changes. Analysis of cell death mode revealed an increase in early apoptotic cells.

Conclusion: Therefore, neonicotinoid insecticides are potentially neurotoxic, reinforcing concerns about human exposure to these commercial formulations.

Keywords: apoptosis; cytotoxicity; imidacloprid; neonicotinoids; reactive species production; thiamethoxam.

Fig. 1

Cell death curves of the neonicotinoid insecticides. MTT reduction (A, C, and E) and the neutral red uptake (B, D, and F) assays were used to evaluate cell viability in differentiated SH-SY5Y cells after 96 h incubation with Actara® (A and B), Much® (C and D) and Evidence® (E and F) at 37 °C. Data are presented as percentage of cell death relative to the negative controls. Three independent experiments were performed with each concentration tested in three replicates within each experiment.

Fig. 2

ROS and RNS production in differentiated SH-SY5Y cells after incubations with the neonicotinoid insecticides for 96 h at 37 °C. Results are expressed as percentage control ± SEM (n = 3 independent experiments run in three replicates). Statistical comparisons were made using one-way ANOVA/Bonferroni post-hoc test (^**P < 0.01; ^***P < 0.001; ^****P < 0.0001 vs control). DMSO, dimethyl sulfoxide; TBHP, tert-butyl hydroperoxide.

Fig. 3

TMRE inclusion in differentiated SH-SY5Y cells after incubations with the neonicotinoid insecticides for 96 h at 37 °C. Results are expressed as percentage control ± SEM (n = 3 independent experiments run in three replicates). Statistical comparisons were made using one-way ANOVA/Bonferroni post-hoc test (^****P < 0.0001 vs control). DMSO, dimethyl sulfoxide; CCCP, carbonyl cyanide 3-chlorophenylhydrazone.

Fig. 4

Annexin V/PI staining of differentiated SH-SY5Y cells after incubations with the neonicotinoid insecticides Actara® (A), Much® (B) and Evidence® (C) for 96 h at 37 °C. Cells were divided in: Early apoptotic (annexin V+/PI–), late apoptotic (annexin V+/PI+) and necrotic (annexin V–/PI+) cells. Results are expressed as percentage cell population ± SEM (n = 3 independent experiments). Statistical comparisons were made using one-way ANOVA/Bonferroni post-hoc test (^*P < 0.05; ^**P < 0.01; ^***P < 0.001; ^****P < 0.0001 vs control). DMSO, dimethyl sulfoxide; PI, propidium iodide.

Fig. 5

Bax protein expression in SH-SY5Y cells incubated with Actara®, Much® or Evidence® for 96 h at 37 °C. Data are expressed as mean ± SEM.