Cannabigerol (CBG, CAS 25654-31-3, purity 99.6%), cannabidiol (CBD, CAS 13956-29-1, purity 98.7%), cannabichromene (CBC, CAS 20675–51-8, purity 99.6%), cannabidivarin (CBDV, CAS 24274-48-4, purity 98.3%), cannabinol (CBN, CAS 521-35-7, purity 99.0%) were obtained from LGC Standards (Augsburg, Germany). Dimethyl sulfoxide (DMSO, CAS 67–68-5) was from Carl Roth (Karlsruhe, Germany). RPMI 1640 medium (Cat. No. R5886), L-glutamine (CAS 56-85-9), sodium pyruvate (CAS 113-24-6), penicillin (100 µg/mL)/streptomycin (1 mM) (Cat. No. P0781), methyl methane sulfonate (MMS, CAS 66–27-3), methanol (CAS 67–56-1, ≥ 99.9%), ethanol (CAS 64-17-5, ≥ 99.9%), cytochalasin B (CAS 14930–96-2), 1,4-diazabicyclo[2.2.2]octane (DABCO, CAS 280-57-9), 4′,6-diamidino-2-phenylindole (DAPI, CAS 28718–90-3), bisbenzimide (Hoechst 33,258, CAS 23491–45-4), FITC-conjugated anti-human IgG secondary antibody (Cat. No. F0132), anti-α-tubulin-FITC monoclonal antibody (Cat. No. F2168), and Tween20 (CAS 9005-64-5) were from Sigma-Aldrich (Steinheim, Germany). Fetal calf serum (Cat. No. AC-SM-0190) was obtained from Anprotec (Bruckberg, Germany). Mutazyme™ S9 mix (Cat. No. 11-405L) was obtained from Trinova Biochem (Giessen, Germany). Vincristine sulphate (CAS 57-22-7) solution was from TEVA (Ulm, Germany). Primary anti-centromere antibody (Cat. No. 15-234) was procured from Antibodies Incorporated (Davis, USA).

Gel Green Nucleic Acid stain (Cat. No. 41003) was obtained from Biotium (Darmstadt, Germany).MACSQuant® reagents (Cat. No. 130-125-753) for flow cytometry were acquired from Miltenyi Biotec (Bergisch Gladbach, Germany).

Human lymphoblastoid cells (TK6) were obtained from Dr. W.J. Caspary, NIEHS, RTP, USA and cultured in RPMI 1640 medium supplemented with 10% (v/v) fetal calf serum, 1% (w/v) l-glutamine, 1% (w/v) sodium pyruvate and 0.4% (w/v) antibiotics (penicillin/streptomycin) in an incubator with 5% CO2 at 37 °C. Cells were sub-cultured three times per week. Cells were seeded in a 6-well plate with 3 mL of medium for each experiment.

Cannabinoid treatment concentrations were chosen according to preliminary data, in the range from non-toxic to moderate toxicity, seen as a reduction of proliferation index. The cells were treated for 4 h with CBG: 5–35 µM, CBD: 5–25 µM, CBC: 5–15 µM, CBDV: 5–40 µM, and cannabinol (CBN): 5–20 µM. Methyl methane sulfonate (MMS) was used as a positive control in a final concentration of 40 µM. The solvent control was DMSO. After 4 h of treatment (following recommendations in the OECD test guideline No. 487) (OECD 2023), the culture medium was renewed, and cells were exposed for 24 h to the cytokinesis inhibitor cytochalasin B (3 μg/mL). Then, cells were harvested, and slides were prepared with cytospin centrifugation. The cells were fixed in ice-cold methanol for at least 2 h. The slides were dried, and Gel Green staining (1:100 dilution in bi-distilled water) was applied for 7 min in the dark. Slides were mounted using 1,4-diazabicyclo[2.2.2]octane (DABCO) and coverslips. The TK6 cells were also treated with CBG 30 µM, CBD 15 µM, CBC 15 µM, CBDV 40 µM, and CBN 15 µM for 4 h with or without the addition of Mutazyme™ 5% S9 mix. Mutazyme™ consists of PB/BNF-induced male Sprague Dawley rat liver S9, which was lyophilized with NADP, D-glucose-6-phosphate, MgCl2/KCl in pH 7.4 sodium phosphate buffer. The S9 mix was dissolved directly in the RPMI-1640 medium and applied at a final concentration of 0.25%. An additional positive control for S9 mix experiments was aflatoxin B1 (5 µM). After the treatment, cells were processed as described above. Each independent experiment was repeated three times. Slides were coded before evaluation. The evaluation was performed with a Nikon Eclipse TE2000 fluorescence microscope. Mononucleated, binucleated, multinucleated, mitotic, and apoptotic cells were evaluated in 1000 cells, micronuclei were scored in 1000 binucleated cells on two slides each, and the mean was calculated. The cytokinesis-block proliferation index (CBPI) was calculated with the following formula:

$$\frac.\text+2\times \text.\text+3\times \text.\text}.\text+\text.\text+\text.\text}.$$

The cells were treated for 6 h with CBG 30 µM, CBD 15 µM, CBC 15 µM, CBDV 40 µM, and CBN 15 µM. The positive control was vincristine sulphate 0.0121 µM, and the solvent control was DMSO. For the experiments with or without the S9 mix (final concentration 0.25%), TK6 cells were treated with either CBDV 40 µM or vincristine sulphate 10 ng/mL for 6 h. DMSO with or without S9 mix was used as control. Further procedure was as described for micronucleus experiments, except with direct harvest after cannabinoid treatment without the incubation period with cytochalasin B. Two slides were evaluated with a Nikon Eclipse 55i fluorescent microscope for each experimental condition. The independent experiments were conducted three times.

The cells were counted in categories apoptosis, interphase, prophase, metaphase, anaphase-telophase, disturbed metaphase, and disturbed anaphase-telophase in 1000 cells. The mitotic index was calculated as a sum of the number of cells in prophase, metaphase, anaphase-telophase, disturbed metaphase, and disturbed anaphase-telophase in 1000 cells. Disturbed metaphase and disturbed anaphase-telophase were defined as deviating from the usual appearance of mitoses. Disturbed mitoses were assessed in 100 mitotic cells per slide. Representative sample images of mitotic disturbances are shown in Fig. 2.

Representative images of disturbed metaphase (a, b) and anaphase-telophase (c, d) in TK6 cells. The cells were stained with Gel Green

For kinetochore analysis, cells were treated for 4 h with CBG 30 µM, CBD 15 µM, CBC 15 µM, CBDV 40 µM, and CBN 15 µM. The positive controls were MMS 40 µM, and vincristine sulphate 0.0121 µM; the solvent control was DMSO. After 4 h of treatment, the culture medium was renewed, and cells were exposed for 24 h to the cytokinesis inhibitor cytochalasin B (3 μg/mL). Then, cells were harvested, and slides were prepared with cytospin centrifugation. The cells were fixed in ice-cold methanol for 2 h. Kinetochores were stained overnight with undiluted primary anti-centromere antibody at 37 °C followed by 1:20 diluted FITC-conjugated secondary antibody at 37 °C for 2 h. Nuclei were counterstained with Hoechst 33,258 (5 µg/mL, 3 min). The experiment was performed one time. Per compound, 200 micronuclei were evaluated for kinetochore signal presence (Fig. 3). The evaluation was performed with a Keyence BZ-X800 microscope. The slides used in kinetochore evaluation were also evaluated for the number of induced micronuclei as described in the above paragraph cytokinesis‑block micronucleus assay.

Representative image of kinetochore analysis. Nuclei (blue) were stained with Hoechst 33,258, and kinetochores (green) with primary anti-centromere and FITC-conjugated secondary antibodies. The arrow indicates the micronucleus with the kinetochore signal

For tubulin visualization, cells were treated with CBG 30 µM, CBD 15 µM, CBC 15 µM, CBDV 40 µM, and CBN 15 µM. The positive control was vincristine sulphate 0.0121 µM, and the solvent control was DMSO. Cells were treated for 6 h and then harvested. Slides were prepared with cytospin centrifugation. The cells were fixed in ice-cold methanol for 2 h. Following fixation, cells were permeabilized with 0.1% Tween20 in 1 × phosphate-buffered saline (PBS) for 5 min. Next, the anti-α-tubulin antibody was applied in 1:50 dilution. Antibody was diluted with 5% FCS in 1 × PBS. The antibody was incubated in a humid chamber at 4 °C for 24 h. Afterwards, the antibody was removed by washing slides twice with 0.5% Tween20 in 1 × PBS for 5 min. Nuclei were counterstained with Hoechst 33,258 (5 µg/mL, 3 min). The visualization was performed with a Keyence BZ-X800 microscope.

The cells were treated with CBG 30 µM, CBD 15 µM, CBC 15 µM, CBDV 40 µM, and CBN 15 µM for 6 or 24 h. The positive control was vincristine sulphate in a concentration of 0.0121 µM for 6 h of exposure and 0.0061 µM for 24 h. The solvent control was DMSO. Furthermore, experiments with the addition of S9 mix were performed with CBDV. In these experiments, cells were treated with CBDV (40 µM) with or without 0.25% S9 mix for 6 h. DMSO with or without the S9 mix was used as control, and the positive control was the same as mentioned above. After the treatment, cells were collected, washed twice with phosphate buffered saline (PBS) and fixed with 70% ethanol for 30 min on ice. Following each described step above, cells were centrifuged at 1000 rpm, 5 min, and 4 °C. Lastly, cells were resuspended in 1% bovine serum albumin (BSA) in PBS solution and stained with DAPI in a final concentration of 1 µg/mL for 30 min on ice. The cell distribution in different cell cycle phases was analyzed with MACSQuant® Analyzer 16 Flow Cytometer using a 450/50 nm filter and MACSQuantify™ Software 2.13 (Miltenyi Biotec, Bergisch Gladbach, Germany). For each sample, 20 000 events were analyzed. Four independent experiments were performed for 6 h of treatment without metabolic activation. Three independent experiments were done for 24 h of treatment without metabolic activation and 6 h of treatment with metabolic activation.

All results are expressed as mean ± standard deviation (SD) from at least three independent experiments except kinetochore labelling results, which are from a one-time experiment. The data were analyzed, and the graphic was drawn with GraphPad Prism version 8 (GraphPad Software, LaJolla, USA). The CBMN and mitotic disturbance assay results were analyzed by one-way ANOVA followed by Dunnett's multiple comparisons tests. The cell cycle analysis results were analyzed by two-way ANOVA followed by Dunnett's multiple comparisons test. The statistical assessment was based on comparing results obtained from cells treated with test compounds and those obtained from cells treated with solvent control. The t-test was used for CBMN experiments with or without the S9 mix to determine the statistical differences between the groups after treating the cells with compounds. The multiple t tests were utilized for cell cycle analysis experiments with or without the S9 mix to check the significance between groups after treating the cells with solvent control or compounds. The results were considered significant when the p value was < 0.05. The BMD50 estimation was obtained from the micronucleus data using the EFSA web tool for Bayesian BMD analysis, which uses the R-package [BMABMDR] version 0.0.0.9083 for the underlying calculations.