Neurotoxicity and Developmental Neurotoxicity of Copper Sulfide Nanoparticles on a Human Neuronal In-Vitro Test System

Abstract

Nanoparticles (NPs) are becoming increasingly important novel materials for many purposes, including basic research, medicine, agriculture, and engineering. Increasing human and environmental exposure to these promising compounds requires assessment of their potential health risks. While the general direct cytotoxicity of NPs is often routinely measured, more indirect possible long-term effects, such as reproductive or developmental neurotoxicity (DNT), have been studied only occasionally and, if so, mostly on non-human animal models, such as zebrafish embryos. In this present study, we employed a well-characterized human neuronal precursor cell line to test the concentration-dependent DNT of green-manufactured copper sulfide (CuS) nanoparticles on crucial early events in human brain development. CuS NPs turned out to be generally cytotoxic in the low ppm range. Using an established prediction model, we found a clear DNT potential of CuS NPs on neuronal precursor cell migration and neurite outgrowth, with IC50 values 10 times and 5 times, respectively, lower for the specific DNT endpoint than for general cytotoxicity. We conclude that, in addition to the opportunities of NPs, their risks to human health should be carefully considered.

Keywords: DNT; NT-2; NTera-2; migration; nanoparticles; neurite outgrowth.

Figure 1

Three endpoints assessed on NT-2 cultures in vitro. (a) General cytotoxicity was assessed by measuring the fluorescence of resazurin converted into resorufin by mitochondrial activity of undifferentiated NT-2 cells. (b) NT-2 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 migrated a distance of ~400 µm on average into this hole. The white arrows indicate direction of cell migration. (c) Neurite growth was assessed by cultivating dissociated NT-2 cultures after 2 weeks of exposure to retinoic acid (containing ~20–40% neurons) and measuring ß-tubulin type III labelled neurites (red) after 24 h. Green lines: example measurements of two neurites, 46 µm and 76 µm long. Scale bars: 400 µm (B) and 50 µm (C). (modified after [17], http://creativecommons.org/licenses/by/4.0/ accessed on 14 April 2024).

Figure 2

Example cultures of undifferentiated NT-2 cells treated for 18 h with 4.1 ppm (a,b) or 111 ppm nanoparticles (c,d). Phase contrast images in b and d, combined with DAPI fluorescence of nuclei. Scale bar 100 µm.

Figure 3

General cytotoxicity concentration–response curve: each value is the average ± S.E.M. of five independent experiments normalized to the control. The IC50 (dotted lines) was 36.99 ppm.

Figure 4

Example of NT-2 cells after 9 days of differentiation and 44 h exposed to test compounds; nuclei labelled with DAPI. Cells were exposed to (a) the cell culture media only (cont), (b) 100 µM cytochalasin D (Cyt D), (c) 50 µM Y-27632, (d) 3.125 ppm nanoparticles, (e) 12.5 ppm nanoparticles, and (f) 50 ppm nanoparticles. Scale bar 200 µm.

Figure 5

Migration assay concentration-response curves: each value is the average ± S.E.M. of three independent experiments normalized to the control. Blue circles: general cytotoxicity (resazurin); red circles: migration distance. Asterisks indicate significant differences (** p < 0.01, *** p < 0.005) from controls; (# p< 0.05, ### p < 0.005) indicate significant differences between viability and migration at the specified concentration. IC50 values are indicated by dotted lines.

Figure 6

Neurite outgrowth: example of NT-2 neurons after 14 days of differentiation and 24 h exposed to test compounds, immunolabelled against ß-tubulin Type III. Cells were exposed to (a) the cell culture media only (cont), (b) 100 µM cytochalasin D (Cyt D), (c) 50 µM Y-27632, (d) 3.125 ppm nanoparticles, (e) 50 ppm nanoparticles, and (f) 200 ppm nanoparticles. Scale bar 50 µm.

Figure 7

Neurite outgrowth concentration-response curves: each value is the average ± S.E.M. of three independent experiments normalized to the control. Blue circles: general cytotoxicity (resazurin); red circles: neurite length/cell. Asterisks (*) indicate significant differences (* p < 0.05, ** p < 0.01, *** p < 0.005) from controls; (## p < 0.01) indicate significant differences between viability and neurite outgrowth at that concentration. IC50 values are indicated by dotted lines.