**3. Discussion**

The aim of this study was to (1) examine the protective effect of resveratrol against Cr(VI)-induced genotoxic damage in vivo and, (2) explore the possible protective pathways of resveratrol at the time of greatest induction of genotoxic damage by CrO3 (48 h). The evaluations were carried out on the same peripheral blood samples, in which the protection from genotoxic damage (MN) was studied. The pathways explored were adduct repair 8-OHdG levels, antioxidant system GSH levels, and enzymatic activities of SOD, GPx, and CAT. Our findings showed that the administration of resveratrol 4 h prior to exposure to CrO3 reduced the frequency of MN induced by this metal in Hsd:ICR male mice. Similarly, an approximation of the possible pathways involved in the protection of genotoxic damage induced by CrO3 was achieved.

To evaluate the genotoxic damage attributed to Cr(VI), a dose of 20 mg/kg CrO3 was administered intraperitoneally (ip). The 20 mg/kg dosage was based upon a previous study, in which this dose induced the formation of MN in the peripheral blood of mice [19,26]. Cr(VI) detoxification is relatively fast (no more than 48 h) when administered ip, in contrast to the effects observed with long-term oral and inhalation exposure to Cr(VI) [27]. Although the ip route is an artificial exposure route, it is useful for detecting genotoxic damage in short-term protocols, such as the MN assay, when testing compounds with potential clastogenic properties. Similarly, it is a more sensitive and direct route than inhalation or oral exposure [28,29]. Thus, a short-term protocol using the ip route of administration was selected to examine direct genotoxic damage induced by CrO3. Resveratrol bioavailability studies, which have described peak plasma concentrations from approximately 1 to 6 h after treatment [30], also support the use of a short-term protocol to assess genotoxic damage.

The increase in MN is an indication of the genotoxic effects exerted by Cr(VI). According to the guidelines of the Organization for Economic Cooperation and Development (OECD) and the Environmental Protection Agency (EPA), a substance or compound that induces more than 4 MN/1000 PCE should be considered a genotoxic agent [29,31]. Our results are consistent with the genotoxic damage reported for Cr(VI) compounds and particularly CrO3 [26,32], as increases greater than 5 MN were observed at all times evaluated. Administration of resveratrol 4 h before exposure to CrO3 reduced these frequencies of MN in vivo. When evaluating the levels of 8-OHdG in blood plasma at 48 h after CrO3 treatment, no significant alterations in adduct repair were detected. Notably, Maeng et al. [33] reported that inhalation of 18 mg/m<sup>3</sup> of sodium chromate resulted in significantly elevated 8-OHdG levels in the lungs after 1 week. However, after 2 weeks of exposure, this dose produced no significant differences in pulmonary 8-OHdG levels, with full recovery after 3 weeks. Maeng et al. [33] also demonstrated that inhalation of higher sodium chromate levels did not significantly alter pulmonary 8-OHdG levels. Similarly, Thompson et al. [34] noted that the in vitro genotoxicity of Cr(VI) is primarily oxidative in nature at low concentrations. They observed that 8-OHdG reaches non-cytotoxic concentrations at 24 h in cell cultures treated with different doses of sodium dichromate. It is conceivable that ip administration of 20 mg/kg CrO3 might be too high to significantly affect 8-OHdG levels in the blood, in agreement with in vivo findings of Maeng et al. [33] and in vitro observations of Thompson et al. [34]. However, it should not be ruled out that Cr(VI) might reduce the levels of protein expression initiating DNA mismatch repair by inhibiting the hMLH1 and hMLH2 genes and the 8-oxoguanine DNA glycosylase1 (OGG1) repair enzyme involved in base excision repair (BER) [35–37]. Mice treated with resveratrol prior to CrO3 showed an elevation in 8-OHdG levels. There are the following two possible explanations for these results: (1) resveratrol activated repair mechanisms that counteract oxidative damage in DNA, and/or (2) resveratrol contributed to the elimination of 8-OHdG

adducts formed by the oxidative damage. The 8-OHdG levels are known to be related to the balance between oxidative DNA damage and the rate at which it is repaired [25]. Yan et al. [38] observed in vitro that resveratrol activates the BER pathway, increasing the expression of OGG1. Further, Mikuła-Pietrasik et al. [39] noted that resveratrol enhanced the activity of the repair enzyme OGG1 in senescent human cells. In our study, resveratrol provided approximately 50% protection against genotoxic damage from CrO3 at all evaluation times. This effect might be attributed to the antioxidant properties of resveratrol, which enable this substance to interact with H2O2 and O2 • and •OH radicals [40]. Previously, Leonard et al. [41] demonstrated in vitro that resveratrol scavenged •OH in JB6 cells exposed to Cr(VI). In a previous study in vivo with (-)-epigallocatechin-3-gallate (EGCG), García-Rodríguez et al. [42] reported that 8-OHdG levels returned to control levels when EGCG and Cr(VI) were co-administered, contrasting with the findings of this study. They also found that co-administration of EGCG and Cr(VI) decreased the magnitude of MN increase compared to Cr(VI) alone [42], similar to the effect observed in this study with resveratrol. Hence, it is possible that when resveratrol was administered in combination with CrO3, the repair mechanisms were enhanced by this polyphenol, contributing to a reduction in MN levels.

In the group treated with resveratrol and CrO3, MN frequencies were reduced by 60, 51, and 46% at 24, 48, and 72 h, respectively. However, the reduction at 72 h was no longer significant. This may be due to the pharmacokinetics of CrO3. It has been reported that Cr(VI) compounds might be excreted within 48 h of exposure [27,43]. Hence, the greatest damage to DNA occurs during that period. Another possibility is that the micronucleated PCE induced over 24 h matured into NCE by 72 h, such that these were not quantified at that time. When leaving the bone marrow, PCE degrades ribosomal RNA in 24 h [44]. In other studies, this same trend was also observed in the reduction of MN at 72 h after administration of Cr(VI) [19,45].

The evaluations of SOD, GPx, CAT, and GSH activities were performed in peripheral blood samples obtained at 48 h because this did not require sacrificing the animals, which was necessary to continue with the evaluation of MN kinetics in the same individuals. Further, in vitro studies showed that resveratrol's antioxidant properties may neutralize oxidative capacity in human erythrocytes [46], and SOD and GSH play important roles in the antioxidant system of erythrocytes [47]. In addition, altered functions of extracellular antioxidants may be assessed by the evaluation of antioxidant molecules in plasma [48]. SOD and GSH measurements were carried out in erythrocytes while CAT and GPx were carried out in plasma.

In the group treated with CrO3, there was a decrease in SOD activity accompanied by an elevation in GPx and CAT activities, which is consistent with previous findings. Both in vitro [49] and in vivo studies demonstrated the effects of oral [50] and ip [42] administration of Cr(VI) compounds on endogenous antioxidants, such as activities of SOD, CAT, GPx, heme oxygenase-1 (HO-1) and levels of GSH. Matés [51] proposed that SOD plays an important role as a first-line antioxidant defense enzyme by catalyzing the dismutation of O2 • to form H2O2, which is subsequently reduced to H2O by GPx and CAT. The decrease in SOD activity observed in the group treated with CrO3 might be related to its depletion by reacting with the O2 • radicals that are generated in excess during reduction to Cr(III). Meanwhile, the H2O2 generated by the activity of SOD may be increased due to the activity of GPx and CAT that was observed in this group. Regarding GSH levels, no significant changes were observed with CrO3 treatment, suggesting that reduction of Cr(VI) was not primarily mediated by this pathway. In this sense, in vivo studies noted that the reduction of Cr(VI) compounds is predominantly via nicotinamide adenine dinucleotide phosphate (NADPH) and other reducing agents such as ascorbate, cysteine, lipoic acid, fructose, and ribose [15]. In the group treated with resveratrol and CrO3, GSH levels were significantly decreased. GSH is a primary antioxidant molecule that plays a fundamental role in reducing cellular oxidative stress. GSH might act in the following different ways: (1) directly as an electron donor by eliminating O2 •, (2) through

GPx catalysis by reducing H2O2 levels, or (3) by forming complexes with detoxifying enzymes such as glutathione S-transferase (GST) [52]. GSH, GPx, and GST are some of the major antioxidant defense systems that scavenge ROS [53]. Resveratrol was found to induce phase II detoxification enzymes in in vitro and in vivo systems [54], and to increase GST activity in cultured aortic smooth muscle cells [55]. Further, several investigators have shown that polyphenols elevate GSH levels and stimulate the transcription of genes that are relevant for the synthesis of endogenous antioxidants, thus counteracting oxidative stress [56]. On the other hand, in this same group (resveratrol + CrO3), the activities of GPx and CAT were restored, and SOD activity increased. Data suggest that resveratrol might counteract CrO3-induced oxidative stress by an indirect antioxidant effect related to the regulation of the endogenous antioxidant system. In in vitro studies, Yao et al. [57] observed that resveratrol protected against oxidative damage induced by sodium sulfate dextran, while Chen et al. [8] found that polyphenols diminished damage mediated by sodium arsenite. In both cases, evidence indicated that the observed protection was related to increased SOD activity. SOD is a phase 2 enzyme that can be activated through the Nrf2/Keap1 signaling pathway. Nrf2 is a fundamental sensor of oxidative stress that plays a central role in the regulation of phase 2 antioxidant and detoxifying enzymes and related proteins [58]. Zhuang et al. [59] found that resveratrol regulates p-Nrf2 levels in a dose-dependent manner. This suggests that resveratrol attenuates the oxidative state, probably by activating the Nrf2 signaling pathway, which in turn elevates SOD activity. Resveratrol was also found to maintain the cellular redox balance by enhancing the activity of antioxidant enzymes, including HO-1, CAT, GPx, and SOD, in rat arterial endothelial cells [60]. Banu et al. [10] showed that 10 mg/kg resveratrol protected against potassium dichromate-induced oxidative stress in rat ovarian tissue by enhancing the activities of GPx, CAT, SOD, peroxiredoxin, and thioredoxin, and by lowering the concentration of H2O2. Therefore, it is possible that resveratrol also removed H2O2 generated by SOD activity, which reduced the need for GPx and CAT activation in the group treated with resveratrol and CrO3. Nevertheless, it is important to keep in mind that the endogenous antioxidant system is dynamic, and thus, it is possible that our results may depend upon the evaluation time (48 h), the dose of resveratrol used, and the experimental model.

Although the PCE/NCE ratio is included in the MN assay as an indicator of cytotoxicity [29], no marked changes in the PCE/NCE ratio were observed in any of the treated groups in this study. These results need to be interpreted with caution, since when toxicity occurs during erythropoiesis, activation of cell division mechanisms may mask the effects on the PCE/NCE ratio [61]. For this reason, cytotoxicity was assessed by analyzing cell viability using the AO/EB differential staining technique, which allows us to distinguish between viable and non-viable cells according to the integrity of the membrane [45]. When cell viability, apoptosis, and necrosis were determined, treatment with CrO3 significantly increased the numbers in total, early and late apoptotic cells, as well as necrotic and nonviable cells. These results corroborate the cytotoxicity reported for Cr(VI) compounds [26,27,45]. ROS generation and DNA damage induced by Cr(VI) exposure might play an essential role in cytotoxicity and the apoptotic signaling pathway [62]. It has been proposed that apoptosis induction is mediated by DNA damage sensors that directly activate p53 through proteins such as DNA-dependent kinase (DNA-PK) or indirectly through mutated ataxiatelangiectasia (ATM) and ATM-Rad3 (ATR) with Chk1 or Chk2 [63]. It has also been reported that apoptosis induction may be mediated through p53-independent pathways such as cleaved caspase 3 and cytochrome C [10,64]. However, when polyphenol was administered 4 h prior to CrO3 in mice, a significant increase in early apoptotic cell number was observed compared to the group treated with CrO3 alone. This effect was masked when early and late apoptotic cells were summed, as late apoptotic cells also showed a significant decrease in this group. Hence, it is possible that the enhanced induction of early apoptotic cells following combined CrO3 resveratrol treatment may contribute to the elimination of cells containing Cr(VI)-induced DNA (MN) damage. Although there are no apparent studies of the effects of resveratrol on Cr(VI)-induced apoptosis pathways, resveratrol was reported to induce apoptosis as a mechanism of elimination of damaged cells in cancer cell lines [4]. Further, alterations in the expression of the Bcl2 protein, the loss of mitochondrial function, the release of cytochrome c, and the activation of caspases trigger the response for the activation of apoptosis [65]. Mirzapur et al. [66] reported that in breast cancer cells, resveratrol elevated the levels of the Bcl2/Bax protein, as well as the expression of p53 genes and caspases 3 and 8. Therefore, based upon these observations and the results reported in the present study, there is clearly a need to conduct studies that aim to reach a more detailed understanding of how resveratrol interacts with proteins such as p53, DNA-PK, ATM, ATR, Bax, Bcl2, caspases (3 and 8), among others. Indeed, these studies may greatly contribute toward understanding the mechanisms by which polyphenols such as resveratrol might contribute to the elimination of cells with genotoxic damage induced by compounds with carcinogenic potential such as Cr(VI).

The administration of resveratrol reduced the frequency of MN induced by CrO3 and resveratrol treatment itself did not produce DNA damage (MN induction). The reduction in GSH and elevation in apoptotic cell number with both treatments (resveratrol + CrO3), as well as increases in SOD, GPx, CAT, and 8-OHdG (the latter non-significant) with resveratrol alone, suggest a toxic effect. In in vitro and in vivo studies, it has been observed that resveratrol exhibits biphasic effects (antioxidant and prooxidant). Meira-Martin et al. [67] considered that the increases in SOD and SOD/CAT activity observed in vitro with different doses of resveratrol are generated to maintain the cellular redox balance. Hence, it has been proposed that its prooxidant activity contributes to the activation of the endogenous antioxidant system [68]. Sinha et al. [69] found that the prooxidative effects of resveratrol are associated with the generation of the O2 • radical, H2O2, and a complex mixture of semiquinones and quinones. However, in this study, no marked effects on viable cell numbers were observed in the group treated with resveratrol and CrO3, and this group exhibited a significant decrease in necrotic cell frequency, suggesting that polyphenols diminished the cytotoxicity produced by CrO3. Other in vivo studies also noted that resveratrol diminished the toxicity induced by metals, such as arsenic [70], copper, and zinc [71], contributing to the balance of the cellular redox system and reducing the expression of proinflammatory cytokines. On the other hand, the administration of resveratrol alone significantly decreased total and early apoptotic cells when compared to its control, suggesting that resveratrol alone does not induce toxicity and that it reduced the potential toxic effect of the vehicle (30% ethanol). Although ethanol is a less toxic polar vehicle than other vehicles such as dimethyl sulfoxide (DMSO) [72], ad libitum administration (11%) was shown to increase serum ROS in treated mice for 60 days [73]. Based on our results, it is suggested to extend these studies by using more diluted doses of resveratrol and even administering it in repeated doses, to reduce the possible toxic effects and improve the protection against the genotoxic damage observed in the present study.

#### **4. Materials and Methods**

#### *4.1. Chemicals and Reagents*

Cr(VI) [CrO3, purity grade 99.9%; CAS 1333-82-0], 3,4 ,5-trihydroxy-trans-stilbene [resveratrol, purity grade ≥ 98%; CAS 501-36-0], AO [CAS 10127-02-3], and EB [CAS 1239-45-8] were obtained from Sigma Chemical Co. (St. Louis, MO, USA).

#### *4.2. Animals*

A group of 25 adult male Hsd:ICR mice (8–12 weeks old, 28–35 g) were used in the experiment. The animals were obtained from Harlan® (Mexico City, CDMX, Mexico) at the "Facultad de Química, Universidad Nacional Autónoma de México-UNAM" and acclimated for two weeks prior to initiating the experiments. During the acclimation period, the groups of five mice were kept in a plastic cage at a controlled room temperature (22 ± 2 ◦C) with a 12-h light-dark cycle (the lights came on at 7:00 a.m. and went off at 7:00 p.m.). Mice had free access to food (Purina-Mexico®, Mexico City, CDMX, Mexico; small rodent chow) and water. Considering that in previous studies there were no differences between males

and females in the genotoxic effects of CrO3 administered by ip injection [16,26,45], this study was carried out using only male mice, in accordance with guidelines for the testing of chemicals (mammalian erythrocyte micronucleus test) of the OECD and the EPA [29,31].

The mice were randomly divided into five groups of five individuals each. Two control groups were used (C1: mice treated ip with sterile distilled water and C2: mice treated with 30% ethanol by gavage) because the CrO3 solution was prepared by dissolving the compound in water, whereas resveratrol was dissolved in 30% ethanol. The resveratrol group was treated with a single dose of 50 mg/kg by gavage, and the CrO3 group was treated with a single dose of 20 mg/kg ip. The last group received combined resveratrol and Cr(VI) treatments (resveratrol + CrO3); the mice were treated with resveratrol at 50 mg/kg by gavage 4 h prior to CrO3 ip injection (20 mg/kg).

The assessment was carried out on peripheral blood obtained from the tail vein since this does not require animals to be sacrificed.
