Cytotoxic, genotoxic, and oxidative stress-related effects of lysergic acid diethylamide (LSD) and phencyclidine (PCP) in the human neuroblastoma SH-SY5Y cell line

Analytical standards of the tested drugs LSD and PCP (Lipomed; Arlesheim, Switzerland) were solved in methanol (Merck, Darmstadt, Germany) to obtain the concentration of 1 mg/mL. Other reagents and chemicals used for experiments were bought from Sigma-Aldrich (Steinheim, Germany).

SH-SY5Y cells (ECACC 94030304, passage numbers 17–23) were obtained from a certified cell-bank, the European Collection of Authenticated Cell Cultures (ECACC), through Sigma-Aldrich. Until treatment, they were cultivated at 37 °C in a 5 % CO₂ atmosphere in DMEM F12 medium containing 15 % (v/v) foetal bovine serum, 2 mmol/L glutamine, 1 % (v/v) penicillin/streptomycin, and 1 % (v/v) non-essential amino acids. The medium was replaced every few days.

Three separate experiments for LSD and PCP were conducted by applying the same experimental design.

One day before each experiment, the cells were seeded in 96-well plates at a density of 20,000 cells/well. On the day of the experiment, the cells were exposed to the tested drugs at concentration range of 0.39–100 μmol/L for 24 h according to experimental schedule presented in Table 1. Drug concentrations were selected based on available literature (8–11).

During treatment, cell cultures were grown at 37 °C in a 5 % CO₂ atmosphere. Untreated cells were used as negative control.

After the treatment, the cells were washed with phosphate-buffered saline (PBS, pH 7.4) and analysed with the MTS assay, alkaline comet assay, and biochemical assays for oxidative stress parameters.

The cytotoxic properties of the tested drugs were studied by measuring mitochondrial succinate dehydrogenase activity with the MTS detection reagent assay (CellTiter 96^® AQueous One Solution Cell Proliferation Assay, Promega, Madison, WI, USA) according to the test protocol reported by Zandona et al. (12). Briefly, the treated cells were rinsed with PBS buffer. Then, 100 μL of DMEM F12 and 20 μL of MTS reagent were pipetted in each well, and cultures incubated for up to 3 h. After that, absorbance was read on a plate reader (Infinite M200PRO, Tecan Austria GmbH, Salzburg, Austria) at 492 nm. Data were pooled from three experiments with duplicate or triplicate evaluations. Results are reported as percentages (mean ± standard deviation) of viable cells with respect to control.

Given that for reliable genotoxicity assessment viability has to be over 70 % (13), for further LSD toxicity assessment we selected the concentration of 6.25 μmol/L, which resulted with the highest cytotoxicity in this experiment. The effects of PCP were further studied at 3.13 μmol/L, as this concentration produces comparable cytotoxicity with LSD, and falls among the values relevant for human exposure in vivo (10, 11).

DNA damaging effects were studied using the alkaline comet assay, whose effectiveness in monitoring DNA damage in neural cells has been well established (14). The extent of DNA damage in single cells was assessed with the standard alkaline comet assay (15) with minor adjustments. Agarose microgels were prepared on fully frosted microscope slides (Surgipath^®, Cambridgeshire, UK). For each experimental point, duplicate slides were prepared. The first layer of gel consisted of 0.6 % normal melting point (MP) agarose. It was then covered with (i) a mixture of 0.5 % low MP agarose and cell samples (V=15 μL of suspension containing 10⁵ cells per slide) and (ii) the top layer of 0.5 % low MP agarose.

To prepare positive control slides, we exposed microgels with untreated cells to 50 μmol/L hydrogen peroxide on ice for 10 min. Hydrogen peroxide was selected, as it induces extensive DNA damage, detectable by the comet assay. Generally, such use of an established genotoxic substance as positive control is recommended to achieve positive response comparable to that of tested substances (16).

Prepared slides were all processed further in the same way. Following polymerisation, microgels were immersed overnight in a lysis buffer (pH=10) composed of 100 mmol/L Na₂EDTA, 2.5 mol/L NaCl (Kemika, Zagreb, Croatia), 10 mmol/L Tris-HCl, 1 % N-lauroylsarcosine sodium salt, 1 % Triton^TM X-100, and 10 % dimethyl sulphoxide (Kemika). Gels were then placed for 20 min in a denaturation buffer (pH >13) composed of 300 mmol/L NaOH (Kemika) and 1 mmol/L Na₂EDTA. The same buffer was used for electrophoresis, which was run at 25 V, 300 mA, and 4 °C for 20 min. Slides were then rinsed three times with 0.4 mol/L Tris-HCl buffer (pH=7.5). Microgels stained with ethidium bromide (20 μg/mL) were observed under a fluorescence microscope (Olympus BX50, Tokyo, Japan) at 200× magnification. Fifty randomly selected comets were scored for each of the two microgels using the Comet Assay IV^TM software (Instem-Perceptive Instruments Ltd., Suffolk, Halstead, UK). As the experiment was carried out in triplicate, altogether 300 comets for each sample were measured. The degree of DNA damage was defined using two comet descriptors: tail DNA% and tail length (presented in micrometres).

Malondialdehyde (MDA) is a reactive end-product of polyunsaturated fatty acid peroxidation. Its levels were determined using the method described by Khoschsorur et al. (17). Treated cells were washed twice in the Krebs-Heneseleit buffer, centrifuged at 248 g for 5 min, and lysed by sonication on ice. Cell lysates were then resuspended in 250 mL of the Krebs-Heneseleit buffer. The reaction mixture consisted of 25 μL of cell lysate (or standard, i.e. 2.5 μmol/L 1,1,3,3-tetraethoxy propane), 225 μL of ultrapure water (18 MΩ, Milli-Q Gradient water system, Thermo Scientific Smart2pure 3 UV/UF, Thermo Electron LED GmbH, Langenselbold, Germany), 375 μL of phosphoric acid (0.44 mol/L), and 125 μL of thiobarbituric acid (TBA, 42 mmol/L, Sigma Chemicals, St. Louis, MO, USA) and kept in a hot water bath for 30 min.

The analysis was performed using a HPLC instrument with UV detector (Shimadzu Corporation, Kyoto, Japan). The guard column and analytical column were C-18 reverse-phase (LiChrospher, Merck) with 5 μm particles (4.0x4.0 mm and 4.0x125.0 mm, respectively).

The mobile phase was 50 mmol/L KH₂PO₄ (Merck) and methanol (60:40, v/v, pH 6.8, Sigma Chemicals). The injection volume was 50 μL, flow-rate 1 mL/min, while absorbance at fluorescence detector was set to 527 (λex) and 550 nm (λem). The retention time of MDA was about 2.5 min. MDA concentration was calculated from the calibration curve using the instrument’s software (Shimadzu LCsolution) and expressed as nmol/mL.

The content of intercellular reactive oxygen species (ROS) was determined using a fluorescent dye 2’,7’-dichlorodihydrofluorescein diacetate (DCFH-DA). After this compound enters a cell, it is first converted into a non-fluorescent product that, in the presence of peroxyl, hydroxyl, and other ROS, is further oxidised to the fluorescent product 2’,7’-dichlorodihydrofluorescein (DCF) (18).

Cells were cultivated in the same medium and manner as for other assays, at 37 °C in a CO₂ incubator. The measurements were done in triplicate in dark-sided 96-well microplates. 100 μL of cell suspension containing 10⁴ cells was pipetted into each well. To assess the amount of intercellular ROS, the culture medium was discarded, and the cells rinsed with 100 μL of PBS and incubated with 200 μL of 20 μmol/L DCFH-DA at 37 °C for 30 min. The dye was then discarded, and the cells rinsed again with 100 μL of PBS and treated with the same concentrations of drugs as in other assays. Fluorescence was recorded on a multilabel plate reader (Victor3^™, Perkin Elmer, Waltham, MA, USA) at the excitation wavelength of 485 nm and emission wavelength of 535 nm. The obtained fluorescence arbitrary units were converted into percentages of control values.

Intracellular glutathione (GSH) was measured with the fluorimetric assay based on the use of fluorogenic monochlorobimane probe (mBCl). Reaction with GSH results in the formation of a fluorescent product in an amount proportional to the content of GSH (19).

Cell cultures in dark-sided 96-well plates were prepared in the same medium and conditions as for other assays. Each well contained 100 μL of cell suspension with 10⁴ cells. Cells were treated with the same concentrations of drugs as in other assays, followed by a 4-hour incubation at 37 °C in a CO₂ incubator. After rinsing with PBS, cells were incubated with 200 μL of 20 μmol/L mBCl in PBS at 37 °C for 30 min. The concentration of GSH in cell samples was measured with a Victor3^™ multilabel plate reader (Perkin Elmer) at the excitation wavelength of 355 nm and emission wavelength of 460 nm. The obtained fluorescence arbitrary units were then converted into percentages of control values. The measurements were done in triplicate.

Antioxidant enzyme activities were measured in triplicate in the cells seeded in 96-well plates and recorded on a plate reader (Tecan Infinite M200PRO, Tecan Austria GmbH). Treated cells were harvested with a rubber policeman and then centrifuged to remove the lysate buffer at 2,000 g and 4 °C for 10 min.

Glutathione peroxidase (GPx) activity was measured with the Assay Kit No.703102 (Cayman Chemical Company, Ann Arbor, MI, USA) according to producer’s instructions (20). After a sequence of reactions with this kit, the decline in absorbance of NADPH (as it oxidises into NADPH⁺) at 340 nm is directly proportional to GPx activity in the sample. One unit of activity corresponds to the amount of GPx needed to catalyse oxidation of 1 nmol of NADPH per minute and per milligram of protein.

Superoxide dismutase (SOD) activity was measured with the Assay Kit No. 706002 (Cayman Chemical Company) according to producer’s instructions (21). The assay uses a tetrazolium salt to detect superoxide radicals produced by xanthine oxidase and hypoxanthine. One unit of activity corresponds to the amount of SOD needed to produce 50 % dismutation of the superoxide radical. SOD activity is expressed as U/mg of protein.

Catalase (CAT) activity was measured with the Assay Kit No. 707002 (Cayman Chemical Company) according to producer’s instructions (22). The method relies on enzyme reaction with methanol in the presence of H₂O₂ at an optimal concentration, which yields formaldehyde. Its amount was measured colourimetrically at 540 nm with 4-amino-3-hydrazino-5-mercapto-1,2,3-triazole (Purpald^®, Sigma-Aldrich) used as the chromogen. One unit of activity corresponds to 1 nmol of formaldehyde per minute and per milligram of protein.

Protein content was determined with the QuantiPro™ BCA Assay Kit (Sigma-Aldrich) according to producer’s instructions. The assay relies on the production of a Cu²⁺-protein complex under alkaline conditions, accompanied by a reduction of the Cu²⁺ to Cu⁺. The extent of this reduction is proportional to protein concentration.

Besides descriptive statistics for each experiment, cell viability data, whose means more accurately represent the centre of distribution, were analysed with parametric one-way analysis of variance (ANOVA) and Dunnett’s test using the Prism 8 software (GraphPad Software, San Diego, CA, USA).

Data with non-parametric distribution (whose medians more accurately represented the centre of distribution), such as the comet assay and biochemical assay were analysed with the Mann-Whitney U-test on Statistica version 14. (TIBCO Software, Inc., Palo Alto, CA, USA).

Statistically significance was set at p<0.05.

Table 2 shows SH-SY5Y cell viability after 24 h of exposure to LSD and PCP in the concentration range of 0.39–100 μmol/L. Compared to control, LSD significantly decreased cell viability in the concentration range from 0.78 to 6.25 μmol/L, and PCP in the concentration range from 1.56 to 100 μmol/L.

Results of the alkaline comet assay (Table 3) demonstrate statistically significant increase in tail DNA% and tail length after LSD treatment and significant increase tail length after PCP treatment compared to negative control.

Figure 1 also shows comet frequency distribution in treated and control cells by tail DNA% and length divided into four quartiles. With LSD the number of comets in the <25^th percentile significantly decreased for both tail DNA% and length, while the proportion of those exceeding the 75^th percentile significantly increased compared to control. With PCP, we observed no significant differences in tail DNA% pattern, but the number of comets in the 50^th–75^th and >75^th length percentile increased significantly.

Figure 1

Oxidative stress-related effects determined in SH-SY5Y cells after treatments with 6.25 μmol/L (2.02 mg/L) LSD, 3.13 μmol/L (0.76 mg/L) PCP and negative control are shown in Figures 2–7.

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 2 shows that 24 h treatment with LSD significantly increased MDA level compared to control, while PCP had the opposite effect.

Figure 3 shows a significant increase in DCFA fluorescence with LSD treatment and, therefore, a significantly higher ROS production compared to control.

Figure 4 shows that LSD significantly increased GSH level compared to control, while PCP had the opposite effect.

Both LSD and PCP significantly increased GPx (Figure 5) and SOD activities compared to control (Figure 6). LSD also significantly increased CAT activity, while PCP had the opposite effect (Figure 7).