Copper pyrithione and zinc pyrithione induce cytotoxicity and neurotoxicity in neuronal/astrocytic co-cultured cells via oxidative stress

Abstract

Previous studies on copper pyrithione (CPT) and zinc pyrithione (ZPT) as antifouling agents have mainly focused on marine organisms. Even though CPT and ZPT pose a risk of human exposure, their neurotoxic effects remain to be elucidated. Therefore, in this study, the cytotoxicity and neurotoxicity of CPT and ZPT were evaluated after the exposure of human SH-SY5Y/astrocytic co-cultured cells to them. The results showed that, in a co-culture model, CPT and ZPT induced cytotoxicity in a dose-dependent manner (~ 400 nM). Exposure to CPT and ZPT suppressed all parameters in the neurite outgrowth assays, including neurite length. In particular, exposure led to neurotoxicity at concentrations with low or no cytotoxicity (~ 200 nM). It also downregulated the expression of genes involved in neurodevelopment and maturation and upregulated astrocyte markers. Moreover, CPT and ZPT induced mitochondrial dysfunction and promoted the generation of reactive oxygen species. Notably, N-acetylcysteine treatment showed neuroprotective effects against CPT- and ZPT-mediated toxicity. We concluded that oxidative stress was the major mechanism underlying CPT- and ZPT-induced toxicity in the co-cultured cells.

Figure 1

Morphological changes caused by retinoic acid (RA) in a human co-culture cell model. (A) Images of the co-cultured cells captured by phase-contrast microscopy: (left) undifferentiated cells in complete medium, (right) differentiated cells in low serum medium supplemented with RA for 5 days. Scale bar: 100 μm. Red arrows indicate astrocytes. Yellow arrows show some neurites of SH-SY5Y cells. (B) Neuronal markers (nestin, microtubule-associated protein 2 and βIII-tubulin) and astrocyte marker (GFAP) expression as determined by western blot analysis. (C) The images were acquired using a fluorescence microscope. Cells were co-stained with an anti-βIII-tubulin antibody (orange) and an anti-GFAP antibody (green) in the presence of RA. Nuclei were counterstained with Hoechst 33342 (blue). Scale bar: 200 μm. (D) The images were obtained using a fluorescence microscope with a 10× objective lens. Cells were stained with an anti-βIII-tubulin antibody (green) in presence and absence of RA. Nuclei were counterstained with Hoechst 33342 (blue). Scale bar: 100 μm. (E) Bar graph displays the average length of neurites following the treatments with RA for 5 days. ***p < 0.001.

Figure 2

Effects of copper pyrithione (CPT) and zinc pyrithione (ZPT) on cell viability and lactate dehydrogenase (LDH) release in the co-culture model. (A) Structure of CPT and ZPT. (B) Cell viability was determined using the CellTiter 96 Aqueous One Solution Cell Proliferation Assay Kit. Data are expressed as percent of the vehicle control and are presented as means ± standard error of the mean (SEM). Results were obtained from three independent experiments. *p < 0.05, **p < 0.01, and ***p < 0.001. (C) Lactate dehydrogenase (LDH) release was determined by the CytoTox 96 Non-Radioactive Cytotoxicity Assay Kit. The values are shown as a percentage of lysis buffer (maximum LDH release), and data are shown as means ± SEM. Data are representative of triplicate experiments. **p < 0.01 and ***p < 0.001 compared to the control.

Figure 3

Effects of copper pyrithione (CPT) and zinc pyrithione (ZPT) exposure on neurotoxicity. SH-SY5Y and astrocyte co-cultured cells were cultured for 5 days with retinoic acid (RA) and then treated with indicated concentration of CPT and ZPT for 24 h. (A) Immunocytochemical images display cells stained with an anti-βIII-tubulin antibody (green) and Hoechst 33,342 (blue). Scale bar: 100 μm. (B) The quantified values are shown as the percentage of control and error bars represent mean ± standard error of mean (SEM). All data were obtained from three independent experiments. *p < 0.05, **p < 0.01, and ***p < 0.001 compared to the control. (C) Expression of NES, TUBB3, MAP2, GAP43, RET, GFAP, and S100β were determined using quantitative real-time polymerase chain reaction. The results were normalized to β-actin expression. Data are mean ± SEM. *p < 0.05, **p < 0.01, and ***p < 0.001.

Figure 4

Effects of copper pyrithione (CPT) and zinc pyrithione (ZPT) on reactive oxygen species (ROS) generation and mitochondrial function in co-cultured cells. Co-cultured cells were incubated with CPT and ZPT at the indicated concentration for 24 h. (A) Intracellular ROS were measured with a microplate reader using DCF-DA fluorescent dye. Cells treated for 1 h with 20 μM tert-Butyl hydroperoxide solution (TBHP) served as a positive control. Results of at least three independent experiments are presented as the mean ± standard error of mean (SEM). *p < 0.05, **p < 0.01, and ***p < 0.001 versus control. (B) Mitochondrial membrane potential (MMP) was determined using a fluorescence probe, JC-1, as described in Section “Material and methods”. Cells treated for 10 min with 20 μM carbonyl cyanide 3-chlorophenylhydrazone (CCCP) served as a positive control. The results were presented as mean ± SEM. *p < 0.05, **p < 0.01, and ***p < 0.001 versus control.

Figure 5

Protective effect of N-acetylcysteine (NAC) against copper pyrithione (CPT) and zinc pyrithione (ZPT) toxicity in co-cultured cells. The cells were pretreated with NAC at 2 mM for 1 h followed by incubation with CPT and ZPT for 24 h. (A) Cell viability was determined using the CellTiter 96 Aqueous One Solution cell Proliferation Assay Kit. Data of at least three independent experiments are presented as the mean ± SEM. (B) Representative fluorescent images of cells stained with an anti-βIII-tubulin antibody (green) and Hoechst 33342 (blue). Scale bar: 200 μm. (C) Data of triplicates are shown as mean ± SEM. (D) The DCF-DA fluorescence intensity was measured using a microplate reader. Error bars represent the mean ± SEM of three independent experiments. (E) Disruption of the mitochondrial membrane potential (MMP) was detected by JC-1 staining and measured using a microplate reader. The graph was expressed as a percentage compared to the control group, and carbonyl cyanide 3-chlorophenylhydrazone (CCCP) was used for the positive control group. Data are representative of at least three independent experiments. *p < 0.05, **p < 0.01 and ***p < 0.001 compared to the control group. ^#p < 0.05, ^##p < 0.01, and ^###p < 0.001 compared to the CPT- or ZPT-treated group.