The Allium cepa test system

The biological material used in this study, as a plant test system, to assess the effects of the ethanolic extracts, was based on seeds of Allium cepa (2n = 16 chromosomes), obtained from the TopSeed^® (Agristar do Brasil Ltda) trademark, from the same batch and variety (Baia Periforme onions). This plant species is indicated for evaluation in genotoxicological studies (Leme and Marin-Morales, 2009).

The exposures were performed as shown in Figure 1. Initially, 14 Petri dishes containing 100 seeds each were maintained in an incubator at 22 ± 2 °C, until they reached about 0.5 cm. After this stage, four groups were treated differently, using two plates per treatment and per concentration.

Group A: negative control (NC). The seeds and the roots remained exposed to ultrapure water during the whole experiment. Samples were collected every 24 hours for a period of 72 hours of exposure (total of 3 samples).

Group B: positive control (PC). The germinated roots were exposed to a methyl methanesulfonate (MMS - CAS no. 66-27-3, Sigma-Aldrich) solution at a concentration of 4 × 10⁻⁴ M. Three collections were made for this group, concomitantly with the NC collections.

Group C: solvent control (SC). The roots were exposed to two ethanolic solutions with concentrations similar to those found in the extracts (SC-1 = 0.042 μL/mL and SC-2 = 0.21 μL/mL, equivalent to the ethanolic extracts concentration of 0.06 μL/mL and 0.30 μL/mL, respectively). The collection followed the same pattern of the NC and PC groups.

Group D: treatments with the ethanolic extracts. In this group, the roots were individually exposed to the extracts for a period of 24 hours. After this time, a part of the roots was collected, while the remainder was transferred to another Petri dish containing a solution of MMS, at the same concentration as the PC. After another period of 24 hours, a part of the roots was collected and fixed, while the other part was transferred to another Petri dish for a recovery assay in ultrapure water for 24 hours. After the recovery period, the material was also collected and fixed.

Thus, for each treatment assay series, we also performed a collection of the control groups (NC, PC and SC). Two independent experiments were performed, in which the exposures were conducted simultaneously, in duplicate, for each experiment. The biological material collected was fixed by a Carnoy I solution (3 parts of ethanol and 1 part of acetic acid - v/v) and stored in a refrigerator at 4 °C until the processing.

For slide preparation, the procedure described by Bianchi et al. (2011) was followed, in which the previously fixed root tips were washed in distilled water and hydrolyzed in HCl 1N at 60°C for 8 min. The roots were washed in distilled water again and submitted to a Schiff's reaction for 2 h. Next, the meristematic and F1 regions were cut, covered with a coverslip and carefully squashed into a drop of 2% acetic carmine solution.

Ten slides were prepared per treatment, five from each duplicate, in order to evaluate the presence of chromosomal aberrations and micronuclei, taking into account the percentage of occurrence. About 500 cells from each slide were analyzed, totalling around 5,000 cells per treatment. This same procedure was followed for the F1 regions of the respective meristems. The slides were analyzed by light microscopy (Carl Zeiss Standard Binocular Microscope) at 400 x magnification.

Cytotoxic and anticytotoxic effects were evaluated by the mitotic index (MI) calculation, as follows: MI=(total number of cells on division/total number of observed cells)x100 (Leme and Marin-Morales, 2009). Genotoxic and antigenotoxic effects were assessed by the observation and counting of the several types of chromosomal aberrations (CA) seen in meristematic cells, like nuclear buds, binucleated cells, polyploidy cells, chromosomal adherence, C-metaphases, chromosomal bridges, chromosomal loss and breakage, and multipolar anaphases (Leme and Marin-Morales, 2009). Mutagenic and antimutagenic potentials were evaluated by the observation and counting of micronuclei (MN) present on meristematic and on F1 cells (Leme and Marin-Morales, 2009). Antigenotoxic and antimutagenic activities were assessed by the analysis of the percentage of damage reduction for each treatment with EEGP and EEBD, respectively, by the following formula: Reduction (%) = [(a - b)/(a - c)]x100 (where: a = number of damaged cells in the PC; b = number of damaged cells in each treatment; c = number of damaged cell in the SC). Examples of alterations in the A. cepa test can be observed in Figure 2.

The results obtained were submitted to a D’Agostino & Pearson statistical normality test. As the results did not pass the normality test, we used the non-parametric test of Kruskal-Wallis, followed by the Dunn's multiple comparison tests, with the significance level of p ≤ 0.05.

In order to facilitate the understanding of the results, they will be presented according to the type of substance used in the treatments:

Pre-treatment: based on the first collection, this was important to verify the possible cytotoxicity, genotoxicity and mutagenicity induced by the ethanolic extracts;

Clastogenic treatment: after the pre-treatment, this was performed to evaluate if the ethanolic extracts could protect the A. cepa cells against the damages induced by MMS. This was important to assess the anticytotoxicity, antigenotoxicity and antimutagenicity;

Recovery assay: after the clastogenic treatment, the remaining roots were kept in ultrapure water for 24 hours. This is recommended to assess the possible residual effects of the ethanolic extracts on cellular protection, i.e., to evaluate if the extracts could exert their effects only when the cells are being exposed or if the extracts could have an extended activity.