Chemicals

Low melting point agarose (LMPA) and normal melting point agarose (NMPA) were obtained from Gibco (Paisley, UK). Inorganic salts, dimethyl sulfoxide (DMSO), methanol, propidium iodide, hydrogen peroxide, triton X-100, trizma base, bovine serum albumine (BSA), cyclophosphamide, cytochalasin B, Dulbecco’s phosphate-buffered saline (DPBS), fetal calf serum (FCS), trypsin–EDTA, Na 2 -EDTA, 4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid (HEPES), trypan blue and cyclophosphamide (CP) were purchased from Sigma-Aldrich (Steinheim, Germany).

Test compounds

Cannabidiol (CBD, CAS 13956-29-1, purity 99.95%) was obtained from LGC Standards GmbH (Germany) and cannabidivarin (CBDV, CAS 24274-48-4, purity 99.80%) from Sigma-Aldrich (Milan, Italy). Both compounds were dissolved in methanol.

Cultivation of cell lines (HepG2 and TR146)

The human hepatoma cell line (HepG2) was provided by F. Darroudi (Department of Toxicogenetics, Leiden University Medical Centre, the Netherlands). The cells were grown in Eagle’s Minimal Essential Medium (EMEM, Sigma-Aldrich, Steinheim, Germany) supplemented with 1.0 mM sodium pyruvate (MNP medium) and 10% FCS. The fifth to eighth passages from stock cultures (in liquid nitrogen) were used for the SCGE and MN experiments.

The human cell line TR146 which is derived from buccal epithelial tissue (Rupniak et al. 1985) was obtained from J. G. Rheinwald (Dermatology Institute of Boston, MA USA). The cells were cultivated in Dulbecco’s modified Eagle Medium (DMEM, Sigma-Aldrich, Steinheim, Germany) with 10% FCS. The cells were stored in liquid nitrogen. The fourth to the sixth passage were used for the genotoxicity experiments.

Both cell lines were cultivated under standard conditions (37 °C, humidified atmosphere, 5% CO 2 ). The media were changed every 2–3 days. When the cultures had reached confluency, the cells were washed with DPBS, detached with trypsin/EDTA, centrifuged and sub-cultured.

Measurements of cytotoxic effects

The viability of the cells was determined with a CASY® cell counter (Schärfe-System GmbH, Reutlingen, Germany). This method is based on the determination of electric potential differences (Lindl et al. 2005). Briefly, cells (2.0 × 105 cells/well) were seeded in 24-wells plates (Becton, Dickinson and Company, NJ, USA) in media which contained different concentrations of CBD (0.22–162 µM) and CBDV (0.66–162 µM) for 3 h or 24 h. In all experiments, solvent controls and positive controls were included. The cells were detached with trypsin–EDTA, centrifuged (200g, 5 min, 21 °C) and suspended in 1.0 mL medium. 50.0 µL of these suspensions were transferred to CASY-cups (OLS OMNI Life Science GmbH & Co. KG, Bremen, Germany). For each experimental point, two independent experiments were performed and means ± standard deviations were calculated. Additionally, we tested the viability of the cells after exposure to the test compounds with the trypan blue exclusion technique (Lindl and Bauer 1994).

Single cell gel electrophoresis (SCGE) assays (standard conditions)

The experiments were conducted according to the protocol of Tice et al. (2000) under alkaline conditions. Only cultures with a viability ≥ 80% were evaluated in SCGE assays.

The indicator cells (2.0 × 105 cells/well) were transferred into 24-well plates which contained 1.0 mL medium with different concentrations of CBD and CBDV. The cells (HepG2) were exposed to the test compounds for 3 h and 24 h (3 h: dose range 0.66–54, 24 h: dose range 0.22–18 µM). TR146 cells were treated with the cannabinoids for 3 h (dose range 2.00–54 µM). In all experiments, solvent controls (methanol) and positive controls (H 2 O 2 , 50 µM) were included. The pellets were resuspended in low melting point agarose (0.5% LMPA). Subsequently, the cells were spread on pre-coated agarose slides (1.5% NMPA) and lysed in the dark at 4 °C for at least 60 min. After 30 min of unwinding under alkaline conditions (pH > 13), electrophoresis was carried out for 30 min (300 mA, 1.0 V/cm, at 4 °C); neutralization was performed twice for 8 min. Air-dried slides were stained with propidium iodide (10 µg/mL). Subsequently, the percentage of DNA in the tails was measured by use of an image analysis system (Comet IV, Perceptive Instruments Ltd., Burry St. Edmunds’, UK). For each experimental point, two slides were prepared and 50 nuclei were evaluated randomly on each slide. Two independent experiments were performed.

In experiments with rat liver homogenate (S9), 10 µL S9 mix was added to the inoculation mix (final protein concentration 30 mg/mL). MUTAZYME™ rat S9 mix (10%) was purchased from TrinovaBiochem GmbH (Giessen, Germany). MUTAZYME™ consists of Aroclor 1254-induced male Sprague Dawley rat liver S9 which was lyophilized with NADP, d-glucose-6-phosphate, MgCl 2 /KCl in pH 7.4 sodium phosphate buffer. The mixtures were incubated for 3 h (37 °C; shaking 250 rpm). Subsequently, the cells were washed and processed as described above. Two independent experiments were performed. For each experimental point, two slides were prepared and 50 nuclei were evaluated randomly from each slide.

Single cell gel electrophoresis (SCGE) assays with lesion-specific enzymes

The impact of the drugs on the formation of oxidized DNA bases was monitored in additional experiments with lesion-specific enzymes. Formamidopyrimidine DNA glycosylase (FPG) and endonuclease III (ENDO III) were purchased from Sigma-Aldrich (Steinheim, Germany). To define the optimal concentrations of the enzymes, calibration experiments were carried out before the main experiments [for details see Collins et al. (1997), data not shown].

The cells (HepG2) were exposed to the test compounds as described above. The experiments with lesion-specific enzymes were conducted according to the protocol of Collins and Dusinska (2002).

After lysis, the slides were washed for 8 min twice with enzyme reaction buffer (40 mM HEPES, 0.1 M KCl, 0.5 mM Na 2 EDTA, 0.2 mg/mL BSA, pH 8.0). Subsequently, the nuclei were treated either with 50 µL of the enzyme solutions or with the enzyme buffers. The incubation time for experiments with FPG was 30 min and for Endo III 45 min at 37 °C, respectively. After the treatment, electrophoresis was carried out under standard conditions (30 min, 300 mA, 1.0 V/cm, at 4 °C, pH > 13). After electrophoresis, the slides were processed and evaluated as described above. Two independent experiments were performed. For each experimental point, two cultures were set up. From each culture, two slides were prepared and 50 cells were evaluated from each slide.

Cytokinesis-block micronucleus (CBMN) assays with HepG2

The experiments were conducted as described by Koller et al. (2014). Briefly, 5.0 × 105 cells/well were seeded in 6-well plates with 3.0 mL medium and allowed to attach overnight. Subsequently, the medium was removed after washing with DPBS. The cells were treated with different concentrations (0.07–2 µM) of the test compounds in serum-free medium for 3 h. Cyclophosphamide (final concentration 500 µg/mL) was used as a positive control. After treatment of the cells with the drugs for 3 h, they were washed with PBS. Subsequently, they were incubated with cytochalasin B (3.0 µg/mL) to block cytokinesis and DMEM (with 10% FCS) for 27–28 h. Then, the cells were washed, trypsinized and harvested. Slides were prepared with the cyto-centrifugation method (Fenech 2007). After drying, they were stained with Diff Quick (Dade Behring, Deerfield, IL, USA) and fixed with Entellan (Sigma-Aldrich, Steinheim, Germany).

Per experimental point, two cultures were made. Four slides were prepared and 2000 cells were evaluated. Different endpoints were scored namely, mono-nucleated, binucleated (BN) and multi-nucleated cells as well as the rates of binucleated cells with MN (BN–MN), the total number of MN in binucleated cells (MNi), nuclear buds (Nbuds), and nucleoplasmatic bridges (NPBs). The cytokinesis-block proliferation indices (CBPI) were calculated with 500 cells according to the formula CBPI = [M1 + 2M2 + 3(M3 + M4)]/N (N is the total number of scored cells), M1–M4 refers to the number of cells with one to four nuclei (OECD 2016). The toxicity of the compounds was indirectly assessed by the assumption that a CBPI of 1.0 corresponds to 100% cytotoxicity (OECD 2016). Five concentrations of each drug were used to determine the CBPI values. Two independent experiments were performed; per experimental point, four slides were prepared and 2000 cells were evaluated. In agreement with OECD guideline #487 (OECD 2016), only doses causing less than 60% cytotoxicity were analyzed with regard to formation of nuclear anomalies. Early necrotic cells, characterized by pale cytoplasm and presence of many vacuoles, and late necrotic cells, identified by loss of cytoplasm and damaged nuclear membranes, were scored according to the protocol of Fenech (2007). Apoptotic cells were identified morphologically by changes in the chromatin structure and by nuclear fragmentation (Fenech 2007).

Statistical analyses

All results were analyzed with the GraphPad Prism 5 software system (LaJolla, CA, USA). The data from the SCGE experiments and from the MN assays are presented as means ± SD. The results of CBMN and SCGE assays (under standard conditions and after treatment with lesion-specific enzymes) were analyzed by one-way ANOVA followed by Dunnett’s multiple comparisons test. The t test was used for experiments with/without S9 in TR146 cells to calculate the statistical differences between the groups after the treatment of the cells with both compounds. Differences were considered as significant when the p values were ≤ 0.05.

All statistical calculations are based on comparisons between results which were obtained with cells which had been treated with the test compounds and results which were obtained with corresponding solvent controls.