## 4 Discussion
However, they reported reduced methylation in MutL homolog 1 (MLH1) and MutS homolog 2 (MSH2) genes, which are involved in DNA mismatch repair mechanism. This study suggested for the first time that Cur may exert an antigenotoxic action through the regulation of epigenetic mechanisms. Our results support the use of Cur and EA as antigenotoxic agents to be considered for the prevention of OS-associated genotoxicity. A therapeutic use of Cur has been investigated in several pathological conditions where OS plays a contributive role. In cancer, it exerts an antiproliferative effect, acting as a chemo-preventive agent [74], while, in atherosclerosis, it prevents platelet aggregation by inhibiting thromboxane [75]. Its antioxidant properties have been reported to be beneficial also in neurodegenerative pathologies [76], liver fibrosis [77], diabetes [78], and autoimmune diseases [79]. Importantly, animal studies revealed that Cur supplementation can reduce chronic alcohol and nicotine-induced toxicity [80; 81]. Similarly, an _in vitro_ study revealed that EA protects against nicotine-induced cellular DNA damage and lipid peroxidation [82]. Animal studies investigated the use of EA to counterbalance the negative effects of chemotherapeutic agents for cancer therapy. Both [PERSON] et al. and [PERSON] et al. reported normalized semen parameters, antioxidants concentration and levels of lipid peroxidation, when adiramycin and cyclophosphamide treatments were associated with EA, respectively [83; 84]. In a placebo-controlled double blind trial, cyclists drinking beverages supplemented with EA showed lower levels of carbonyls and 8-OHdG, both markers of OS, after intensive exercise [85]. Despite encouraging results, our study is not free of limitations. Although an _in vitro_ approach is useful to understand the mechanism of actions, it is difficult to forecast effects due to molecular interactions between cells in different tissues or long-term exposure, as in an in vivo system. Moreover, it is difficult to compare the concentration tested with those likely to be introduced through normal nutrition (_curcuruma longa_ 1-2 \(\mu\)g/g), as well as to establish the optimal concentrations to be used for therapeutic purposes starting from _in vitro_ experiments. In fact, in an in vivo system, Cur is subjected to low absorption, poor bioavailability and rapid metabolism. Therefore, the determination of the pharmacokinetic profile of Cur appears difficult due to low circulating concentrations [86]. However, the safety of the substance is maintained in vivo even after the administration of very high doses [87]; in addition, other _in vitro_ studies have demonstrated its efficacy at concentrations ranging from 5 to 50 \(\mu\)M [49; 88]. Similarly, EA is poorly bioavailable and non-absorbable, despite being a very small molecule. In fact, it passes unaltered to the intestinal tract, where it undergoes extensive metabolism by the intestinal microbiota providing undithins (UROs), that are more bioavailable and are much better absorbed than EA. Even when consumed in very large quantities (800 mg of extract), the maximum concentration of EA in plasma was reported to be very low and not sufficient to produce significant beneficial effects [89]. The activity of the human microbiota is difficult to reproduce in animal models and cannot be easily studied and controlled _in vitro_. Hence, we had the same difficulties with EA to compare the concentration tested with those likely to be present through normal nutrition and used therapeutically. In this study we used only one concentration of EA and Cur, but a variety of assays with the primary goal of studying different antigenotoxicity endpoints. Therefore, our future goal will be to evaluate the effects of different EA and Cur concentrations for dose-response experiment, including at increasing exposure times. We cannot exclude that higher concentrations may have a negative influence on zebrafish physiology.