## 4 Discussion  
The antioxidant power of EA and Cur is well known, so the novelty of our research was to assess the compounds' ability to counteract DNA damage caused by a known genotoxic agent (benzene), by using different methodologies in an experimental _in vitro_ model of zebrafish blood cells. Our results confirm the genotoxicity of benzene and highlight EA and Cur as anti-genotoxic molecules, able to counteract ROS-mediated DNA damage. We observed that DNA damage and apoptotic rate were increased when zebrafish blood cells were incubated with benzene. Benzene is a toxic chemical whose mutagenic effect has been studied for decades [32; 33; 34; 35]. Benzene and its metabolites, such as 1,4-benzoquinone and hydroquinone, were described as cytotoxic agents able to induce DNA strand breaks in hamster ovary cells [34]. In human sperm cells, exposure to benzene resulted in reduced sperm motility and vitality, along with increased DNA denaturation [36]. In 2015, [PERSON] et al. showed that hydroquinone (HQ), an important metabolite of benzene which is abundant in cigarette smoking, significantly reduced the expression of multidrug resistance mutation (MDR1) gene, encoding a membrane transporter, and inhibited the activation and nuclear translocation of the transcription factor NF-kB in mesenchymal stem cells derived from bone marrow [32]. This resulted in the induction of apoptotic pathways and reduced cellular vitality. Particularly, benzene cytotoxicity is mediated by the induction of OS [36]. [PERSON] et al. demonstrated that cells incubation in benzene resulted in increased ROS generation and apoptotic rate, along with upregulated expression of OS-related genes [35]. In the presence of noxious oxygen species, antioxidants play an important role in counterbalancing such effect and preserving the physiological redox balance, exerting an anti-genotoxic effect [10; 37]. In this regard, several antioxidant compounds have been identified in plants or food as having the power to counteract the impact of genotoxic agents on DNA [38; 39]. In this study, Cur was chosen for investigation as it acts as antioxidant but also upregulates the expression of transcription factors involved in the response to OS and other endogenous antioxidants, such as superoxide dismutase (SOD), catalase and glutathione (GSH) [40; 41; 42; 43]. EA was selected because its antioxidant and anti-genotoxic potential has  
Figure 4: Changes in percentage of genome template stability (y-axis) in zebrafish blood cells DNA at different exposure times (x-axis). Benzene—BZ; Ellagic acid—EA; curcumin—Cur; negative controls—NC. Letters correspond to diverse statistical significances (ANOVA); _a:_\(p<0.05\) in comparison with NC; _b:_\(p<0.05\) in comparison with BZ exposure. been pointed out by several studies, along with its anti-inflammatory and anti-pathogens properties [16, 44, 45, 46, 47]. EA and Cur tested concentrations were chosen based on preliminary tests and previous literature data [13, 37, 48, 49]. Our data showed that EA and Cur were not harmful for zebrafish blood cells _in vitro_ for the time and concentration investigated. Specifically, no variation was observed in comparison with the negative control at all timepoints analyzed in terms of cytotoxicity by trypan blue, DNA integrity by comet assay and RAPD-PCR and apoptotic rate by diffusion assay. However, the comet assay measures strand breaks, which are not always specific of OS, so in future experiments we plan to incorporate a formamidopyrimidine DNA glycosylase into the incubation assay, which converts oxidized purines into breaks, and is therefore more specific for oxidative damage [50]. We observed that incubation of zebrafish blood cells in EA nullifies the impact of benzene on all the above-mentioned outcomes at all timepoints analyzed, exerting a relevant antioxidant and anti-genotoxic effect. EA is a phenolic molecule, which is abundant in specific plants, mushrooms and food sources [51, 52].