*2.4. Effect of Resveratrol and CrO3 on Apoptotic and Necrotic Cells*

Apoptotic and necrotic cells as well as cell viability were evaluated using differential acridine orange/ethidium bromide (AO/EB) staining (Figure 5A). The dual fluorochrome assay is capable of distinguishing between viable and nonviable cells based on membrane integrity. When cells are still viable, they keep the plasma membrane intact, allowing only AO to intercalate into DNA, which causes the nucleus to fluoresce green (Figure 5A(i,iii)). However, in nonviable cells, membrane integrity is lost, causing, ethidium bromide (EB) to also intercalate into DNA, making the nucleus fluoresce red since EB overwhelms AO staining (Figure 5A(ii,iv)). The color of the nucleus depends on the viability of the cell, not the state of the nucleus. Early apoptotic cells that have intact membranes but in which the DNA has begun to fragment still exhibit green nuclei because the EB cannot enter the cell, but chromatin condensation is visible as bright green patches in the nuclei (Figure 5A(iii)). As the cell progresses through the apoptotic pathway and membrane blebbing begins to occur, EB permeates the cell, producing a red-stained cell. Late apoptotic cells show bright red patches of condensed chromatin in the nuclei (Figure 5A(iv)); this distinguishes them from necrotic cells, which stain uniformly red (Figure 5A(ii)). When comparing the effect of treatments on apoptosis by one-way ANOVA, there was a significant effect of treatment on the frequency of healthy, total, early and late apoptotic cells as well as necrotic cells when compared to their control groups (*p* < 0.0001). Resveratrol reduced the frequency of total and early apoptotic cells compared to control groups (*p* < 0.022 and *p* < 0.015, respectively), while CrO3 induced an increased number of total, early, late apoptotic, and necrotic cells compared to control groups (*p* < 0.0001). In the resveratrol + CrO3 mice, there were fewer late apoptotic and necrotic cells compared to the CrO3 group (*p* < 0.001 and *p* < 0.0001, respectively) and an increase in total and early apoptotic cells compared to the control group (*p* < 0.0001) and the resveratrol group (*p* < 0.0001) (Figure 5B).

**Figure 5.** (**A**) Fluorescent microphotograph (400×) of peripheral blood cells using differential acridine orange/ethidium bromide (AO/EB) staining. (**i**) Healthy cell. (**ii**) Necrotic cell. (**iii**) Early apoptotic cell. (**iv**) Late apoptotic cell. (**B**) Effect of resveratrol and CrO3 on the frequencies of healthy, apoptotic (total, early, and late), and necrotic cells in peripheral blood, evaluated 48 h after treatments. A total of 300 nucleated cells were evaluated in each mouse (*n* = 5 mice/group). Statistical significance was determined using one-way ANOVA followed by Tukey's post-hoc test: <sup>a</sup> *p* < 0.0001 vs. C1; <sup>b</sup> *p* < 0.03 vs. C2; <sup>c</sup> *p* < 0.0001 vs. C2; <sup>d</sup> *p* < 0.0001 vs. resveratrol; <sup>e</sup> *p* < 0.022 vs. C2; <sup>f</sup> *p* < 0.015 vs. C2; <sup>g</sup> *p* < 0.011 vs. CrO3; <sup>h</sup> *p* < 0.034 vs. C2; <sup>i</sup> *p* < 0.02 vs. resveratrol; <sup>j</sup> *p* < 0.001 vs. resveratrol + CrO3; <sup>k</sup> *p* < 0.0001 vs. resveratrol + CrO3. C1, Control 1, vehicle only (distilled water); C2, Control 2, vehicle only (ethanol 30%); CrO3, chromium trioxide.

Table 1 shows the PCE/NCE ratio. These evaluations were performed on the same samples and times used for MN. There were no significant effects in any of the treatments compared to their control groups (C1, C2) or time 0. However, when cell viability was compared in nucleated peripheral blood cells (48 h) using the dual fluorochrome assay, a significant effect of treatment on viable and nonviable cells (*p* < 0.0001) was observed (one-way ANOVA). The dual fluorochrome assay is an indicator of cell metabolism and death caused by cell membrane injury. Viable cells included those with an intact membrane, and thus they exhibited a nucleus fluoresced green by AO intercalation (healthy and early apoptotic cells; Figure 5A(i,iii), respectively). Moreover, nonviable cells included those in which the integrity of the membrane had been lost and that, therefore, presented a nucleus fluoresced red due to the intercalation of the EB (late apoptotic and necrotic cells; Figure 5A(ii,iv), respectively). Treatment with CrO3 increased nonviable cells compared to the control group (*p* < 0.0001), while treatment with resveratrol prior to CrO3 exposure decreased the nonviable cells observed in the group treated with CrO3 alone (*p* < 0.0001). Resveratrol treatment alone had no significant effect on cell viability (Figure 6).


**Table 1.** PCE/NCE ratio in peripheral blood of mice treated with resveratrol and CrO3.

A total of 2000 erythrocytes were evaluated in each mouse (*n* = 5 mice/group). C1, Control 1, vehicle only (distilled water); C2, Control 2, vehicle only (ethanol 30%). CrO3, chromium trioxide; PCE, polychromatic erythrocytes; NCE, normochromatic erythrocytes.

**Figure 6.** Effect of resveratrol and CrO3 on the frequencies of viable and nonviable cells in the peripheral blood of mice evaluated 48 h after treatment using differential acridine orange/ethidium bromide (AO/EB) staining. Viable cells include healthy and early apoptotic cells. Nonviable cells include late apoptotic and necrotic cells. A total of 300 nucleated cells were evaluated for each mouse (*n* = 5 mice/group). Statistical significance was determined using one-way ANOVA followed by Tukey's multiple comparisons post hoc test: <sup>a</sup> *p* < 0.0001 vs. C1; <sup>b</sup> *p* < 0.0001 vs. resveratrol + CrO3. C1, Control 1, vehicle only (distilled water); C2, Control 2, vehicle only (ethanol 30%); CrO3, chromium trioxide.

The mice in the CrO3 group showed clinical signs of toxicity, including bristling hair, decreased mobility, and loss of appetite. The dose of 50 mg/kg of resveratrol did not exhibit any apparent clinical signs of toxicity. None of the mice exposed to resveratrol, CrO3, or both treatments died.
