Mode of Action of Toxin 6-Hydroxydopamine in SH-SY5Y Using NMR Metabolomics

Abstract

This study used NMR-based metabolomics to investigate the mode of action (MoA) of 6-hydroxydopamine (6-OHDA) toxicity in the SH-SY5Y neuroblastoma cell model. 6-OHDA, a structural analogue of dopamine, has been used to create a Parkinson's disease model since 1968. Its selective uptake via catecholaminergic transporters leads to intracellular oxidative stress and mitochondrial dysfunction. SH-SY5Y cells were treated with 6-OHDA at its IC₅₀ concentration of 60 μM, and samples of treated and untreated groups were collected after 24 h. The endo metabolome was extracted using a methanol-water mixture, while the exo metabolome was represented by the culture media. Further, endo- and exo metabolomes of treated and untreated cells were analysed for metabolic changes. Our results demonstrated significantly high levels of glutathione, acetate, propionate, and NAD⁺, which are oxidative stress markers, enhanced due to ROS production in the system. In addition, alteration of myoinositol, taurine, and o-phosphocholine could be due to oxidative stress-induced membrane potential disturbance. Mitochondrial complex I inhibition causes electron transport chain (ETC) dysfunction. Changes in key metabolites of glycolysis and energy metabolism, such as glucose, pyruvate, lactate, creatine, creatine phosphate, glycine, and methionine, respectively, demonstrated ETC dysfunction. We also identified changes in amino acids such as glutamine, glutamate, and proline, followed by nucleotide metabolism such as uridine and uridine monophosphate levels, which were decreased in the treated group.

Keywords: 6-OHDA; ETC; NMR metabolomics; ROS; SH-SY5Y.

Figure 1

(a) PC1 and PC2 scores plot of the PCA analysis for the endo metabolome of 6-OHDA treated (red) and untreated (blue) cells. (b) Bivariate loadings plot of PC1 for the endo metabolome. The x-axis represents chemical shifts of metabolites on a parts per million (ppm) scale. The y-axis represents loading p-values. Overlayed are the absolute values of p(corr) as a heatmap ranging from 0 (blue) being not correlated to 1 (red) being highly correlated.

Figure 2

(a) PC1 and PC2 scores plot of the PCA analysis for the exo metabolome of 6-OHDA-treated (red) and untreated (blue) groups. (b) PCA bivariate loadings plot of PC1. The x-axis represents chemical shifts of metabolites on a parts per million (ppm) scale. The y-axis represents loading p-values. Overlayed are the absolute values of p(corr) as a heatmap ranging from 0 (blue) being not correlated to 1 (red) being highly correlated.

Figure 3

Impacted pathways in the endo metabolome. The x-axis represents pathway impact, and the y-axis pathway enrichment. Darker circle colors indicate more significant changes of metabolites in the corresponding pathway. The size of the circle corresponds to the pathway impact score and is correlated with the centrality of the involved metabolites.

Figure 4

Impacted pathway in the exo metabolome. The x-axis represents pathway impact, and the y-axis pathway enrichment. Darker circle colors indicate more significant changes of metabolites in the corresponding pathway. The size of the circle corresponds to the pathway impact score and is correlated with the centrality of the involved metabolites.

Figure 5

Control experiment to understand the presence of pyroglutamyl alanine. Black arrow alanine (1.49 ppm), Red arrow pyroglutamyl alanine (1.52 ppm), Blue arrow glutamate (2.33 ppm).

Figure 6

Metabolic pathways of significantly altered metabolites in cells treated with 6-OHDA compared to controls. The left side of each cell refers to the endo metabolome and the right side of the cell refers to the exo metabolome. Green colour denotes higher levels; yellow denotes lower levels. ROS: Reactive Oxygen Species.

Figure 7

IC₅₀ of 6-OHDA. The x-axis represents log concentration in molar and the y-axis represents cell viability in percent.