The effects of ketamine on viability, primary DNA damage, and oxidative stress parameters in HepG2 and SH-SY5Y cells

The analytical standard ketamine, consisting of a mixture of both isomers (1 mg/mL in methanol) was purchased from Lipomed (Arlesheim, Switzerland). Potassium dihydrogen phosphate (KH₂PO₄) was obtained from Merck (Darmstadt, Germany). Ultrapure water (18 MΩ) was obtained from a Barnstead^™ Smart2Pure^™ 3 UV/UF Water Purification System (Thermo Fisher Scientific, Waltham, MA USA). Potassium hydroxide (KOH) and phosphoric acid (H₃PO₄) were purchased from Kemika (Zagreb, Croatia).

Other reagents and chemicals were bought from Sigma-Aldrich (Steinheim, Germany). Staurosporine, well-known inductor of apoptosis, was used as positive control for cytotoxicity experiments. Hydrogen peroxide (H₂O₂), oxidising agent, was used as positive control for other assays.

Cell lines used in the experiments were obtained from the European Collection of Authenticated Cell Cultures (ECACC) (Sigma-Aldrich). Human Caucasian hepatocyte carcinoma HepG2 cells (ECACC 85011430) were grown in Eagle’s Minimum Essential (EMEM) medium containing 10 % (v/v) foetal bovine serum, 1 % (v/v) penicillin/streptomycin, and 1 % (v/v) non-essential amino acids. Human neuroblastoma SH-SY5Y cells (ECACC 94030304) were grown in Dulbecco’s Modified Eagle’s Medium F12 (DMEM F12) containing 15 % (v/v) foetal bovine serum, 2 mmol/L glutamine, 1 % (v/v) penicillin/streptomycin, and 1 % (v/v) non-essential amino acids. All media and supplements were purchased from Sigma-Aldrich. All cells were cultured at 37 °C in a 5 % CO₂ atmosphere, and the medium was changed every few days and passaged according to the manufacturer’s protocol.

Cells for the viability assay were seeded in transparent 96-well plates at a density of 20,000 cells/well one day before the assay. Cells for reactive oxygen species and glutathione measurements were seeded in black 96-well plates at a density of 10,000 cells/well. For other assays they were seeded in transparent 24-well plates at a density of 50,000 cells/well. For the viability assay both cell lines were treated with ketamine concentrations from 0.39 to 100 µmol/L. For DNA damage and cell oxidative status determination ketamine concentrations corresponded to those found in human plasma for analgesia (0.09 mg/L i.e. 0.39 µmol/L), anaesthesia (1.49 mg/L i.e. 6.25 µmol/L), and drug abuse (0.37 mg/L i.e. 1.56 µmol/L) (6). The highest tested concentration of 100 µmol/L was selected based on reports that this ketamine concentration affects calcium signalling, which leads to apoptosis in undifferentiated neural PC12 cells (13).

All treated samples were compared to untreated control cells, based on results obtained from at least three independent experiments (each treatment performed in duplicate or triplicate).

Cytotoxic properties of ketamine were determined by measuring mitochondrial succinate dehydrogenase activity of viable cells following a protocol described elsewhere (14). After treatment with ketamine and incubation with the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) reagent (CellTiter 96^® AQueous One Solution Cell Proliferation Assay, Promega, Madison, WI, USA), the absorbance was read at 492 nm on an Infinite M200PRO (Tecan Group Ltd., Switzerland) plate reader. Results are given as percentages (mean ± standard deviation) of viable cells compared to control (untreated) cells. Considering that a reliable genotoxicity assessment requires that cell viability is over 70 % (15), for further toxicity assessments we selected three concentrations which meet this requirement and which correspond to ketamine concentrations found in human plasma after analgesia (0.39 µmol/L), anaesthesia (6.25 µmol/L), and drug abuse (1.56 µmol/L).

The alkaline comet assay was performed to determine the level of primary DNA damage. Its effectiveness in genotoxicity assessments has been well established (16–18). A detailed procedure for microgel preparation has been described in our previous publication (18). Briefly, randomly selected comets were measured on duplicate slides under an epifluorescence microscope (200× magnification) using the Comet Assay IV^TM software (Instem-Perceptive Instruments Ltd., UK). As the experiment was carried out in triplicate, altogether 600 comets per sample were assessed for tail intensity (DNA% in the comet tail) as an indicator of DNA damage.

Malondialdehyde (MDA) analysis in cell lysates was based on the method reported by Khoschsorur et al. (19). Briefly, after the treatment, cells were rinsed twice in Krebs-Heneseleit buffer, centrifuged at 248 g for 5 min and sonicated on ice. After lysis, cell lysate was resuspended in Krebs-Heneseleit buffer (250 mL). The reaction mixture was prepared by mixing 25 µL cell lysates (or standard, i.e. 2.5 µmol/L 1,1,3,3-tetraethoxy propane) with water (225 µL), phosphoric acid (375 µL; 0.44 mol/L), and thiobarbituric acid (125 µL; 42 mmol/L) and incubating it in boiling water bath at 100 °C for 30 min.

Samples were analysed with a high-performance liquid chromatograph (HPLC) with a UV detector (Shimadzu Corporation, Kyoto, Japan) using the guard and analytical reverse-phase C-18 column (4.0 × 4.0 and 4.0 × 125.0 mm, respectively) with 5 µm particle size (LiChrospher, Merck) as described earlier in detail (18).

Reactive oxygen species (ROS) were determined using a fluorescent dye 2’,7’-dichlorodihydrofluorescein diacetate (DCFH-DA) obtained from Sigma-Aldrich. HepG2 and SH-SY5Y were seeded into black 96-well plates at 10⁴ cells/mL. On the day of the experiment, the medium was removed, cells rinsed with the PBS buffer (100 µL) and incubated with DCFH-DA (200 µL; 20 µmol/L) at 37 °C for 30 min (20). The dye was then washed off, and the cells rinsed again with PBS (100 µL) and treated with either of the three concentrations of ketamine mentioned above.

Fluorescence was recorded (λ_Ex 485 nm, λ_Em 520 nm) on a multilabel plate reader (Victor3™, Perkin Elmer, Waltham, MA, USA). Fluorescence arbitrary units are given as percentages of control cell values.

Intracellular glutathione (GSH) was measured using fluorogenic monochlorobimane (mBCl), as it reacts with GSH to form a fluorescent product in an amount proportional to GSH content (21).

Cell cultures were placed in black-sided, clear-bottom 96-well plates in the same medium and conditions as for other assays. Cells were treated with the same ketamine concentrations as in other assays, followed by a 4-hour incubation at 37 °C. Then, the cells were rinsed with PBS and incubated with mBCl in PBS (200 µL; 20 µmol/L) at 37 °C for 30 min. The fluorescence (corresponding to GSH concentration) in cell samples was measured with a Victor3™ (Perkin Elmer) multilabel plate reader (λ_Ex 355 nm, λ_Em 460 nm). The obtained fluorescence arbitrary units are given as percentages of control values. The measurements were done in triplicate.

Glutathione peroxidase (GPx) activity was measured with the commercially available assay kit No. 703102 (Cayman Chemical Company, Ann Arbor, MI, USA) according to the manufacturer’s instructions (22). Briefly, treated cells were separated by centrifugation (1000 g at 4 °C for 10 min), sonicated in cold buffer (50 mmol/L tris-HCl, pH 7.5, 5 mmol/L ethylenediaminetetraacetic acid (EDTA), and 1 mmol/L dithiothreitol (DTT), and centrifuged again at 10,000 g and 4 °C for 15 min to obtain the supernatant. After a sequence of reactions, the decrease in absorbance due to nicotinamide adenine dinucleotide phosphate (NADPH) oxidation into NADP⁺ directly proportional to GPx activity in the sample was measured at 340 nm using the SpectraMax i3x microplate reader (Molecular Devices, San Jose, CA, USA). 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. GPx activity is expressed as percentage of control values.

Superoxide dismutase (SOD) activity was measured with the commercially available assay kit No. 706002 (Cayman Chemical Company) according to the manufacturer’s instructions (23). Briefly, treated cells were separated by centrifugation (1000 g and at 4 °C for 10 min), sonicated in cold buffer [20 mmol/L N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid (HEPES) buffer, pH 7.2, containing 1 mmol/L ethylene glycol tetraacetic acid (EGTA), 210 mmol/L mannitol, and 70 mmol/L sucrose], and centrifuged again at 1500 g and 4 °C for 5 min to obtain the supernatant. Tetrazolium salt is used for the detection of superoxide radicals produced by xanthine oxidase and hypoxanthine. The inhibition of this reaction was measured at 505 nm using the SpectraMax i3x microplate reader (Molecular Devices). One unit of activity corresponds to the amount of SOD needed to produce 50 % dismutation of the superoxide radical. SOD activity is given as percentage of control values.

Catalase (CAT) activity was measured with the commercially available assay kit No. 707002 (Cayman Chemical Company) according to the manufacturer’s instructions (24). Cells collected after treatment and centrifugation at 1000 g and at 4 °C for 10 min were sonicated in cold buffer (50 mmol/L potassium phosphate, pH 7.0, containing 1 mmol/L EDTA) and centrifuged again at 10,000 g at 4 °C for 15 min to obtain the supernatant. The method relies on enzyme reaction with methanol in the presence of H₂O₂ at an optimal concentration, which yields formaldehyde. As the chromogen 4-amino-3-hydrazino-5-mercapto-1,2,3-triazole (Purpald^®, Sigma-Aldrich) was used, the absorbance was measured at 540 nm using the SpectraMax i3x microplate reader (Molecular Devices). One unit of activity corresponds to 1 nmol of formaldehyde per minute and per milligram of protein. CAT activity is given as percentage of control values.

Cell viability data were analysed with the parametric one-way analysis of variance (ANOVA) and Dunnett’s test using the Prism software (GraphPad Software, San Diego, CA, USA). The comet assay data, which had non-parametric distribution (and whose medians more accurately represented the centre of distribution), were analysed with the Mann-Whitney U-test on Statistica version 14 (TIBCO Software, Inc., Palo Alto, CA, USA). The same software was used for the analysis of biochemical parameters with the Kruskal-Wallis one-way analysis of variance. Statistical significance was set at P<0.05.