Developmental Neurotoxicity of Fipronil and Rotenone on a Human Neuronal In Vitro Test System

Abstract

Pesticide exposure during in utero and early postnatal development can cause a wide range of neurological defects. However, relatively few insecticides have been recognized as developmental neurotoxicants, so far. Recently, discovery of the insecticide, fipronil, in chicken eggs has raised public concern. The status of fipronil as a potential developmental neurotoxicant is still under debate. Whereas several in vivo and in vitro studies suggest specific toxicity, other in vitro studies could not confirm this concern. Here, we tested fipronil and its main metabolic product, fipronil sulfone both at concentrations between 1.98 and 62.5 µM, alongside with the established developmental neurotoxicant, rotenone (0.004-10 µM) in vitro on the human neuronal precursor cell line NT2. We found that rotenone impaired all three tested DNT endpoints, neurite outgrowth, neuronal differentiation, and precursor cell migration in a dose-dependent manner and clearly separable from general cytotoxicity in the nanomolar range. Fipronil and fipronil sulfone specifically inhibited cell migration and neuronal differentiation, but not neurite outgrowth in the micromolar range. The rho-kinase inhibitor Y-27632 counteracted inhibition of migration for all three compounds (EC50 between 12 and 50 µM). The antioxidant, n-acetyl cysteine, could ameliorate the inhibitory effects of fipronil on all three tested endpoints (EC 50 between 84 and 164 µM), indicating the involvement of oxidative stress. Fipronil sulfone had a stronger effect than fipronil, confirming the importance to test metabolic products alongside original pesticides. We conclude that in vitro fipronil and fipronil sulfone display specific developmental neurotoxicity on developing human model neurons.

Keywords: DNT; Differentiation; Migration; NT2; NTera-2; Neurite outgrowth.

Fig. 1

Three DNT endpoints assessed on NT2 cultures in vitro. (a) Neuronal differentiation was assessed by measuring immunofluorescence of a monoclonal antibody against b-tubulin type III. On average, ~ 10% of all DAPI-positive cells (blue) display ß-tub III-immunoreactivity (red) after 9 days in culture under control conditions. (b) NT2 precursor cell migration was measured using the Oris cell migration assay, which creates cell culture monolayers with a circular hole. During 2 days in culture, cells migrate a distance of ~ 400 µm on average into this hole. (c) Neurite growth was assessed by cultivating dissociated NT2 cultures after 2 weeks exposure to retinoic acid (containing ~ 20–40% neurons) and measuring ß-tubulin type III labeled neurites (red) after 24 h. Green lines: example measurements of two neurites, 46 µm and 76 µm long. Scale bars: 50 µm (a, c), 400 µm (b)

Fig. 2

Neurite outgrowth assay. Examples are given for NT2 cells after 2 weeks of differentiation cultivated for 24 h in the presence of test compounds and labeled for neuron-specific ß-tubulin type III (red) and DAPI (blue). Cells without red fluorescence are still undifferentiated. Cells were subjected to (a) cell culture media only, (b) 100 µM cytochalasin D (cytD), (c) 7.8 µM fipronil (fip), (d) 62.5 µM fipronil, (e) 50 µM Y-27632, (f) 2.4 µM rotenone (rot only), (g) 2.4 µM rotenone plus 1.111 mM n-acetyl cysteine (rot + NAC), (h) 2.4 µM rotenone plus 50 µM Y-27632 (rot + Y27632). Scale bar: 100 µM

Fig. 3

Neurite outgrowth assay, concentration–response curves. Each value is the average ± S.E.M. of three independent experiments normalized to the solvent control (0.25% DMSO). Hollow blue circles: general cytotoxicity (resazurin), filled red circles: total neurite length/neuron. Asterisks (*) indicate significant differences (at least p < 0.05) from controls, (#) indicate significant differences between viability and neurite length at that concentration. (N) indicates the highest non-cytotoxic concentration (viability > 90%, dotted horizontal line). a Rotenone reduced neurite outgrowth already at 123 nM, whereas general viability was still at 80% of control at the highest tested concentration (10 µM). b Fipronil reduced both viability and neurite outgrowth, but values below 50% were not reached at the highest concentration tested. c Fipronil sulfone reduced both viability and neurite outgrowth at the same concentrations. d The inhibitory effect of 2.4 µM rotenone on neurite outgrowth was compensated in a dose-dependent manner by the rho kinase inhibitor, Y-27632, with an EC50 of 12.14 µM. e The inhibitory effect of 2.4 µM rotenone on neurite outgrowth could not be alleviated by the antioxidant, n-acteyl cysteine (NAC). f The inhibitory effect of 2.4 µM rotenone on neurite outgrowth could not be alleviated by a second antioxidant, α-tocotrienol

Fig. 4

Neuronal differentiation assay. NT2 cells immunolabeled for ß-tubulin III, counterstained with DAPI after 9 days of differentiation, during last 5 days exposed to (a) DMSO only, (b) 11 nM rotenone, (c) 11 nM rotenone + 370 µM NAC, (d) 15 µM fipronil, (e) 15 µM fipronil + 1.111 mM NAC, (f) 15 µM fipronil sulfone. Scale bar 100 µm

Fig. 5

Neuronal differentiation assay, concentration–response curves. Each value is the average ± S.E.M. of three independent experiments normalized to the solvent control. Hollow blue circles: general cytotoxicity (resazurin), filled red circles: neuron-specific immunofluorescence of ß-tubulin type III after 4 days in cell culture media only, followed by 5 days of test compound exposure. Asterisks (*) indicate significant differences (at least p < 0.05) from solvent controls (a–c), exposure to rotenone only (d) or fipronil only (e), (#) indicate significant differences between viability and ß-tubulin III expression at that concentration. (N) indicates the highest non-cytotoxic concentration (viability > 90%, dotted horizontal line). (a) Rotenone reduced neuronal differentiation already at 1.2 nM, whereas general viability was still at 72% at the highest tested concentration. (b) Fipronil reduced both viability and neuronal differentiation in a dose-dependent manner. At 15.6 µM fipronil, differentiation was reduced to less than 50% and differed significantly from general viability (90%) at this concentration. (c) Fipronil sulfone reduced both viability and neuronal differentiation in a dose-dependent manner. At 7.81 µM, differentiation was significantly reduced to 70%, whereas general viability was still close to 100% at this concentration. (d) The inhibitory effect of 11 nM rotenone on neuronal differentiation could not be alleviated by the antioxidant, NAC, at any tested concentration. (e) The inhibitory effect of 15 µM fipronil was alleviated by the antioxidant, NAC, in a dose-dependent manner (EC50 94.7 µM)

Fig. 6

Migration assay. NT2 cells after 9 days of differentiation and 44 h exposed to test compounds, nuclei labeled with DAPI. Cells were exposed to (a) 0.25% DMSO only (cont) (b) 122 nM rotenone (rot), (c) 62.5 µM fipronil (fip), (d) 62.5 µM fipronil sulfone (fip-sulf), (e) 100 µM cytochalasin D (cyt D), (f) 122 nM rotenone plus 50 µM Y-27632, (g) 50 µM fipronil plus 50 µM Y-27632, (h) 15 µM fipronil sulfone plus 50 µM Y-27632, (i) 50 µM Y-27632, (j) 122 nM rotenone plus 1.111 mM n-acetyl-cysteine (NAC), (k) 50 µM fipronil plus 1.111 mM NAC, (l) 15 µM fipronil sulfone plus 1.111 mM NAC. Scale bar 400 µM

Fig. 7

Migration assay, concentration–response curves. Each value is the average ± S.E.M. of three independent experiments normalized to the solvent control. Hollow blue circles: general cytotoxicity (resazurin), filled red circles: migration distance. Asterisks (*) indicate significant differences (at least p < 0.05) from controls, (#) indicate significant differences between viability and migration at that concentration. (N) indicates the highest non-cytotoxic concentration (viability > 90%, dotted horizontal line). (a) Rotenone reduced migration in a dose-dependent manner (IC50 51.0 nM), whereas general viability was reduced, but remained above 70% at the highest tested concentration. (b) The inhibitory effect of 122 nM rotenone could not be alleviated by the antioxidant, n-acetyl cysteine (NAC), at any concentration. (c) The inhibitory effect of 122 nM rotenone was alleviated by co-application of the rho kinase inhibitor, Y-27632, in a dose-dependent manner (EC50 8.3 µM). (d) Fipronil inhibited migration in a dose-dependent manner. At 15.63 µM fipronil and higher, migration was significantly reduced as compared to both migration of controls, and viability at the same concentration. (e) The inhibitory effect of 50 µM fipronil could be alleviated by the antioxidant, n-acetyl cysteine (NAC), in a dose-dependent manner (EC50 161.1 µM). f The inhibitory effect of 50 µM fipronil could not be alleviated by co-application of Y-27632 at any concentration. (g) Fipronil sulfone inhibited migration in a dose-dependent manner. At 15.63 µM fipronil and higher, migration was significantly reduced as compared to both migration of controls, and viability at the same concentration. (h) The inhibitory effect of 15 µM fipronil sulfone was slightly alleviated by the antioxidant, n-acetyl cysteine (NAC), in a dose-dependent manner (EC50 84.63 µM). At 1.111 µM NAC, cells migrated significantly further than under treatment of 15 µM fipronil sulfone (FS) alone. (j) The inhibitory effect of 15 µM fipronil sulfone was alleviated by co-application of the rho kinase inhibitor, Y-27632, in a dose-dependent manner (EC50 4.22 µM)