Artichoke Induces Genetic Toxicity and Decreases Ethyl Methanesulfonate-Related DNA Damage in Chinese Hamster Ovary Cells

Vegetal extract

The C. scolymus L. leaves used in this work were collected in the city of Gramado (Rio Grande Do Sul/Brazil) in a small farm (Apiquários) where the plants are organically cultivated. After collection, specimens were identified, recorded, and prepared in the herbarium of the Department of Botany of the Lutheran University of Brazil (HERULBRA 4288).

Crude aqueous extracts of leaves (120 g) were prepared by infusion (1:10 plant/solvent) with distilled water at 80°C for 30 min. The infusion was left to stand to cool down at room temperature. After cooling, the filtered extract was frozen and concentrated by lyophilization for 5 days overnight to obtain the crude aqueous extract from leaves (13.7 g, yield: 11.4%) of C. scolymus. In a previous study, our research group demonstrated that the phytochemical profile of C. scolymus identified in the LE included flavonoids, phenolic compounds, and saponins. ²⁹

DNA damage-inducing agent

The alkylating agent EMS (CAS, 62-50-0) was purchased from Sigma Chemical Company. EMS was dissolved in Dulbecco's modified Eagle's medium (DMEM) immediately before use and was used as a positive control in a dose of 350 μM.

Culture conditions

Chinese hamster ovary (CHO) cells were kindly provided by Professor Salvadori (UNESP-Botucatu). Cells were grown as monolayers in plastic culture flasks (75 cm²) in DMEM (Gibco) supplemented with 10% fetal bovine serum (Gibco) and 1% antibiotics (streptomycin and penicillin) at 37°C in an incubator chamber with 5% CO₂.

Cell viability tests

To determine the concentrations to be evaluated for genotoxicity, the trypan blue exclusion assay was carried out using cell samples before they were analyzed using the comet assay. After treatment, cells were tested by trypan blue staining. Prepared solution of 15 μL trypan blue (0.4%) in distilled water was mixed with 15 μL of each cell suspension, spread onto a microscope slide, and covered with a coverslip. In this test, the dead cells take up the dye and appear blue. At least 100 cells were evaluated per treatment. Only doses that presented at least 80% of viability were evaluated in the comet assay.

Cell treatment

To determine the genotoxicity and antigenotoxicity of the different concentrations of the C. scolymus extract, each protocol was performed in duplicate on two different days to ensure reproducibility. Positive (EMS) and negative control (DMEM) groups were also included in the analysis. For the experiments, 1×10⁵ CHO cells were seeded in 24-well plates, incubated for 24 h at 37°C and 5% CO₂ atmosphere, in a complete DMEM, washed with Dulbecco's phosphate-buffered saline (DPBS), and then submitted to one of the following treatments in a serum-free medium: (a) negative control; (b) EMS for 1 h (positive control); (c) C. scolymus extract (0.62, 1.25, 2.5, and 5.0 mg/mL) for 1 h; (d) C. scolymus extract (0.62, 1.25, 2.5, and 5.0 mg/mL) for 24 h, in this treatment EMS (positive control) for 24 h; (e) extract plus EMS for 1 h (simultaneous treatment); (f) C. scolymus extract (0.62, 1.25, 2.5, and 5.0 mg/mL) for 1 h before washing the cells and adding EMS for 1 h (pretreatment with extracts); and (g) EMS for 1 h before washing the cells and adding C. scolymus extract (0.62, 1.25, 2.5, and 5.0 mg/mL) for 1 h (post-treatment with extracts). At the end of the treatments, cells were washed with DPBS at 37°C and trypsinized with 350 μL trypsin. After 5 min, the cells were gently resuspended in a complete medium, and 40 μL of the cell suspension was immediately used for the test.

Treatments c and d were experiments to test genotoxicity, and treatments e, f, and g were the experiments to test antigenotoxicity.

The comet assay

The alkaline single-cell gel electrophoresis or comet assay was performed according to Singh et al. ²⁸ and Munari et al. ³⁰ with minor modifications. Briefly, a base layer of 1.5% normal-melting agarose (CAS:9012-36-6; Invitrogen) was placed on a microscope slide, and 40 μL of CHO test cells suspended in 140 μL 0.5% low-melting agarose (CAS:9012-36-6; Invitrogen) at 37°C was then spread over the base layer. A coverslip was added, and the agarose was allowed to solidify at 4°C for 15 min. After agarose solidification, the coverslip was removed, and the slides were immersed into a lysis solution (89 mL stock solution, 10 mL DMSO, 1 mL Triton X-100—pH 10.0), at 4°C for at least 24 h (lysis of cells to liberate DNA), protected from light.

At the end of this period, the slides were washed with DPBS for 15 min, placed into a horizontal electrophoresis chamber, and left immersed in an alkaline solution (300 mM NaOH, 1 mM ethylenediaminetetraacetic acid, pH 13) for 20 min for DNA unwinding. Electrophoresis was carried out for 20 min at 25 V and 300 mA. After electrophoresis, the slides were submerged in a neutralization buffer (0.4 M Tris–HCl, pH 7.5) for 15 min and fixed in 100% ethanol.

Scoring comets

All slides, including those of the positive and negative controls, were coded before microscopic analysis and scored without knowledge of the code. The slides were stained with 70 μL ethidium bromide ²⁸ and covered with a coverslip. The stained nucleoids were immediately evaluated at 1000×magnification under an Olympus (BX41) fluorescence microscope (excitation filter, 515–560 nm; barrier filter, 590 nm).

For each treatment, the extent and distribution of DNA damage indicated by the comet assay were evaluated, classifying comets for visual inspection, typically into five categories based on the length of migration and/or the perceived relative proportion of the DNA in the tail and size of head (nucleus): 0 representing undamaged cells (comets with no tail) and 1–4 representing increasing relative tail intensities and minor head size. ^{27,30 –33}

In total, 600 cells for each dose in the genotoxicity test (1 and 24 h) as well in the antigenotoxicity test in three protocols were evaluated (pretreatment, simultaneous treatment, and post-treatment).

The total score was calculated according to the formula: \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document} \begin{align*}{ \rm Score} = ( 1 \times n_1 ) + ( 2 \times n_2 )+ ( 3 \times n_3 ) + ( 4 \times n_4 )\end{align*} \end{document}

where n=number of cells in each class analyzed. Thus, the total score ranged from 0 to 400.

The percentage of protective effect of LE was calculated according to Waters et al. ³⁴ using the following formula: \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document} \begin{align*} \% \,{ \rm Reduction} - \frac{\hbox{EMS alone score} - \hbox{LE plus EMS score}} {\hbox{EMS alone score} - \hbox{negative control score}} \times 100\end{align*} \end{document}

Statistical analysis

The results were evaluated by analysis of variance and Dunnet test at P<.05. In the genotoxicity experiments, LE treatments were compared in relation to the negative control. In the antigenotoxic evaluation, treatments were compared to the EMS-positive control to observe the modulatory effects of the C. scolymus LE.

Genotoxicity

Tables 1 and 2 show DNA damage in CHO cells treated with different concentrations of artichoke LE. Cells were exposed to four concentrations of the extract and EMS, for 1 and 24 h. All the experiments were conducted in duplicate, and at least three experiments were performed for each exposure time. In the 1- and 24-h treatments, mean scores of CHO cells treated with all four doses of the extract were statistically different against the respective negative control. These results indicate that the C. scolymus LE induces DNA damage in eukaryotic cells. EMS used as a positive control demonstrated the sensitivity of the comet assay and presented a clear positive outcome at the concentration evaluated.

Antigenotoxicity tests

Table 3 shows the extent of DNA damage in CHO cells exposed to three different concentrations of LE and EMS using three different treatment regimens. DNA migration was clearly enhanced in the presence of EMS, when compared to the negative control. In the pre- and post-treatment, no significant reduction was observed in relation to the EMS-induced DNA damage.

Considering the simultaneous treatment, significant reductions were observed for the three LE doses (0.62, 1.25, and 2.5 mg/mL) when compared to lesions induced by EMS alone. The reduction percentage of damaged cells was 77.2%, 17.0%, and 24.6% for the concentrations of 0.62, 1.25, and 2.5 mg/mL, respectively. These results point out the lack of a dose–response correlation, since the lowest concentration was found to be more protective, whereas an increase in the modulatory effect was not proportional to the increase in the concentrations.