*4.6. Apoptosis and Cell Viability*

To evaluate apoptosis, necrosis, and cell viability, differential acridine AO/EB staining was performed using a technique previously adapted for peripheral blood [45]. Ten μL samples were centrifuged at 4500× *g* for 5 min. The cell pellet was resuspended in 20 μL of AO/EB dye mix and plated on a clean slide. Two slides were prepared per mouse, and the analysis was performed immediately. The assessments were based upon 300 cells per mouse. Apoptotic, necrotic, viable, and nonviable cells were identified using a fluorescence microscope OPTIPHOT-2 (NikonTM; Tokyo, Japan) with blue excitation (480 nm) and a barrier filter emission (515–530 nm) at 40× magnification.

#### *4.7. Statistical Analysis*

Each mouse was considered an independent replicate according to the OECD and EPA guidelines [29,31]. Individual samples were averaged for each experimental group. The MN frequencies, PCE/NCE ratio, viability (viable/nonviable cells), number of apoptotic and necrotic cells, levels of 8-OHdG and GSH, and activities of SOD, GPx, and CAT are expressed as the mean ± standard deviation (SD). The data were checked for normality using the Shapiro–Wilk test. Statistical significance between the groups for MN was determined by using two-way RM-ANOVA because MN depends on two factors (i.e., treatment and time). The treatment is independent, while the evaluations at each time are considered dependent since the samples were obtained from the same mouse. For the other parameters, one-way ANOVA was used because the evaluations depended only on one factor (i.e., treatment). In the analysis of ANOVA, post hoc Tukey multiple comparisons were carried out. GraphPad Prism 8.0 (GraphPad Software, San Diego, CA, USA) was used for all analyses. Differences were considered significant at *p* < 0.05.

#### **5. Conclusions**

Our findings demonstrate a protective effect of resveratrol against Cr(VI)-induced genotoxic damage, by reducing the frequency of MN induced by CrO3 in vivo. Likewise, an approximation of the possible pathways involved in the protection of genotoxic damage induced by these compounds with carcinogenic potential, such as Cr(VI), was achieved. Resveratrol showed effects on the modulation of the endogenous antioxidant system, 8-OHdG adduct repair, and apoptosis when administered 4 h prior to Cr(VI) exposure. These effects suggest that these pathways might be involved in the protection provided by this polyphenol against genotoxic damage induced by Cr(VI). Although resveratrol treatment modified endogenous antioxidant system constituents, the dose of 50 mg/kg alone did not alter MN frequencies, suggesting that it is not related to the induction of DNA damage. In vivo studies using more diluted doses of resveratrol and even administering it in repeated doses, as well as direct evaluations in target organs, could help determine the specific mechanisms, by which resveratrol counteracts Cr(VI)-induced genotoxicity. These studies contribute to the understanding of the potential antigenotoxic value of polyphenols such as resveratrol, and to the exploration of their possible use as chemotherapeutic agents in the prevention and treatment of diseases related to genotoxic damage.

**Author Contributions:** Conceptualization, M.d.C.G.-R.; Methodology, T.N.-M.; Validation, M.d.C.G.- R., V.M.M.-N., A.R.O.-M.; Data Curation, T.N.-M., M.d.C.G.-R., V.M.M.-N. and A.R.O.-M.; Investigation, M.d.C.G.-R., T.N.-M.; Writing—Original Draft Preparation, TN-M.; Writing—Review and Editing, M.d.C.G.-R., S.K. and T.N.-M.; Supervision, M.d.C.G.-R., S.K., A.R.O.-M. and V.M.M.-N.; Project Administration, M.d.C.G.-R.; Funding Acquisition, M.d.C.G.-R. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by DGAPA-UNAM, Support Program for Research and Technological Innovation Projects, PAPIIT-IN224719; IN216122. The National Council of Science and Technology granted a scholarship for postgraduate studies (CONACyT, No. 703847) to Tonancy Nicolás-Méndez.

**Institutional Review Board Statement:** The Bioethics Committee of the "Facultad de Estudios Superiores-Zaragoza, UNAM" approved the experimental conditions and protocols (Code: FESZ/DEPI/363/14; FESZ/DEPI/CE/016/21).

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study are available on request from the corresponding author.

**Conflicts of Interest:** The authors declare no conflict of interest.

**Sample Availability:** Samples of the compounds are not available from the authors.

#### **References**

