*2.1. Effect of Resveratrol on MN Induced by CrO3*

The genotoxic damage caused by Cr(VI) was evaluated using the micronuclei (MN) assay in erythrocytes of peripheral blood using acridine orange (AO)-coated slides. Differential AO staining distinguished polychromatic erythrocytes (PCE) from normochromatic erythrocytes (NCE) because PCE were stained, showing orange fluorescence due to the presence of ribosomal RNA (Figure 1A(i)), while NCE did not stain at all (shadow) (Figure 1A(ii)). The AO also enabled the identification of MN, which exhibited yellow fluorescence due to their DNA content (Figure 1A(iii)). To compare the kinetics of MN induction in treatment groups, data were analyzed by calculating the net induction frequency (NIF) using Equation (1) as follows:

NIF = |MN frequencies measured at *time xi* − MN frequencies measured at *time 0*|/*n* (1)

where *xi* is the evaluation at 24, 48, or 72 h per group, *time 0* is the evaluation at 0 h (before treatment) per group, and *n* is the number of mice per group.

Calculating the NIF enhanced the ability to determine net MN induction by eliminating baseline MN variability among treated groups at *time 0*. Figure 1B illustrates the NIF of MN values for all treatments at 24, 48, and 72 h after administration. Treatments had a significant effect (*p* < 0.0001) on the MN frequencies according to the two-way repeated-measures analysis of variance (RM-ANOVA). In the chromium trioxide (CrO3) group, an increase of about 7, 10, and 5 MN was observed at 24, 48, and 72 h, respectively, which was significantly higher than the control C1 (*p* < 0.001, *p* < 0.0001, and *p* < 0.015, respectively). The group treated with resveratrol and CrO3 (resveratrol + CrO3) had lower MN frequency than the CrO3 only treatment at all times examined and was highest at 48 h (*p* < 0.001), though this reduction was no longer significant at 72 h (*p* > 0.05). However, in this group, the MN frequencies observed at 48 h were significantly different from the control groups (C1, *p* < 0.001; C2, *p* < 0.040) and the group treated with resveratrol alone (*p* < 0.006). There was a significant effect of time on the frequency of MN in the two-way RM-ANOVA (*p* < 0.0001). In the CrO3 group, the frequency of MN increased at 48 h with respect to the initial time (*p* < 0.038) and decreased at 72 h (*p* < 0.004). In the resveratrol + CrO3 group, the MN frequency decreased at 72 h (*p* < 0.009), and values at the initial and final times were similar. Treatment with resveratrol alone did not significantly affect the frequency of MN compared to control group C2 (*p* > 0.05).

**Figure 1.** Effect of resveratrol and CrO3 on the frequency of micronuclei (MN) were evaluated in the peripheral blood of mice. (**A**) Fluorescent microphotograph (1000×) of peripheral blood cells using the AO coating method. Polychromatic erythrocytes (PCE) stain fluorescent orange (**i**), normochromatic erythrocytes (NCE) do not stain at all (shadow) (**ii**), and MN fluoresces yellow (**iii**). (**B**) Data show the MN frequency at 24, 48, and 72 h minus the MN frequency at 0 h (net induction frequency: NIF, see results text). The frequencies of MN in the resveratrol + CrO3 group decreased by 63, 50, and 47% at 24, 48, and 72 h, respectively, compared with those in the CrO3 group. A total of 4000 PCE were evaluated in each mouse (*n* = 5 mice/group). Statistical significance was determined using two-way repeated measures-ANOVA followed by Tukey's post-hoc test. Analysis by treatments: <sup>a</sup> *p* < 0.001 vs. C1, 24 h; <sup>b</sup> *p* < 0.004 vs. resveratrol + CrO3, 24 h; <sup>c</sup> *p* < 0.0001 vs. C1, 48 h; <sup>d</sup> *p* < 0.001 vs. resveratrol + CrO3, 48 h; <sup>e</sup> *p* < 0.001 vs. C1, 48 h; <sup>f</sup> *p* < 0.040 vs. C2, 48 h; <sup>g</sup> *p* < 0.006 vs. resveratrol, 48 h; <sup>h</sup> *p* < 0.015 vs. C1, 72 h; NS *p* > 0.05 vs. CrO3, 72 h. Analysis by time of evaluations: <sup>i</sup> *p* < 0.038, CrO3; <sup>j</sup> *p* < 0.004, CrO3; <sup>k</sup> *p* < 0.009, resveratrol + CrO3. C1, Control 1, vehicle only (distilled water). C2, Control 2, vehicle only (ethanol 30%). CrO3, chromium trioxide. AO, acridine orange.
