The effect of mephedrone on human neuroblastoma and astrocytoma cells

Abstract

Mephedrone is an illegal drug that is used recreationally. Few studies have been conducted to investigate the mechanisms by which mephedrone is harming cells. In this research, we investigated the effect of mephedrone using toxicology coupled with LC-MS/MS based metabolomics in the two CNS derived cell lines. Methods of assessment such as neutral red (NR) assay, dimethylthiazolyl diphenyltetrazolium bromide (MTT), lactose dehydrogenase (LDH) measurement, and morphology were performed to identify the effect on cell viability and to identify the best concentration to be used in a metabolomics study. A concentration of 100 _μM of mephedrone was used in the metabolomic experiment because at this concentration mephedrone had induced several intracellular changes. Although there no clear indicators of cellular damage caused by mephedrone. In astrocytes there was a clear indication that cell membrane function might be impaired by depletion of ether lipids.

Keywords: Glutathione; LC-MS/MS; Mephedrone; Metabolomics.

Fig. 1

MTT assay measured in (A) SH-SY5Y cells and in (B) U-373 cells after 48 h. Results are percentage values (Mean ± SD, n = 6) where 100 % corresponds to control values. Cells treated with mephedrone in culture medium throughout the experiment. Data were analysed using one-way ANOVA followed by Tukey test. * Data were significantly different from their relevant control P value < 0.05.

Fig. 2

Neutral red assay measured in (A) SH-SY5Y cells and in (B) U-373 cells after 48 h. Results are percentage values (Mean ± SD, n = 6) where 100 % corresponds to control values. Cells treated with mephedrone in culture medium throughout the experiment. Data were analysed using one-way ANOVA plus Tukey test. * data were significantly different from their relevant control P value < 0.05.

Fig. 3

Morphology of SH-SY5Y (A and B) and U373 (C and D) cells after 6 days incubation without mephedrone treatment (A and C), and with mephedrone (B and D). Pictures were taken by the Motic AE31 microscope-20 power dry lenses.

Fig. 4

OPLS-DA score plot of neuroblastoma (SH-SY5Y) treated with mephedrone (blue) and non-treated (green). Test stands for treated samples and control stands for non-treated samples.

Fig. 5

Permutation analysis of the OPLSDA model for mephedrone treated and non-treated astrocytoma cells. Validation of the model was performed with 100 permutation tests. R₂ represent the goodness of the fit and Q₂ represent the predictive capability.

Fig. 6

PCA model of the control (brown), mephedrone treated (blue) astrocytes and pooled samples (green).

Fig. 7

OPLS-DA score plot of astrocytoma (U-373) treated with mephedrone (blue) and non-treated (green) without pooled samples. C represents the control (non-treated) samples, and T represents the test (treated) samples.

Fig. 8

Permutation analysis of the OPLS-DA model for mephedrone treated and non-treated astrocytoma cells. Validation of the model was performed with 100 permutation tests. R₂ represent the goodness of the fit and Q₂ represent the predictive capability.

Fig. 9

Extracted ion traces showing similar levels of ATP in treated and untreated extracts but a large reduction of the amount of a major cellular lipid (PC 36:2) in treated astrocytoma cells.

Fig. 10

Lipids in astrocytes sorted by abundance before and after treatment with mephedrone. Red = 30 % of maximum value, yellow = 2 % of maximum value and blue = 0. SM = sphingomyelin, CER = ceramide.

Fig. 10

Lipids in astrocytes sorted by abundance before and after treatment with mephedrone. Red = 30 % of maximum value, yellow = 2 % of maximum value and blue = 0. SM = sphingomyelin, CER = ceramide.