*Epigenetic Studies*

Epigenetics is the study of heritable variations in gene function that cannot be attributed to changes in the sequences of coding DNA. There are causal interactions between genes and their products that give rise to the phenotype. In terms of lifestyle, it is noteworthy that different diets providing, e.g., different fatty acids [193] can modulate genetics through epigenetic changes. Several investigators reported epigenetic variations through the study of the mechanisms by which dietary exposure can have long-term consequences for growth and health. As an example, Mathers et al. developed a model of four Rs (Received', 'Recorded', 'Remembered', and 'Revealed') to explain the mechanism of nutritional epigenomics [194]. Other publications addressed the issue of how diet in pregnancy affects fetal programming [195].

All epigenetic variations are most often investigated by assessing histone modification, DNA methylation, and non-coding RNAs. Histone modifications by methylation, acetylation, ubiquitination, and phosphorylation determine an active or inactive state of chromatin and, thus regulate gene expression. DNA methylation consists of the addition of methyl groups at the 5-position of a cytosine and is frequently part of a cytosine-guanine dinucleotide (CpG). These are clustered in the 5 ends of genes in regions known as "CpG islands." This methylation is associated with the silencing of gene transcription and is a dynamic process that occurs throughout life [196]. Table 2 includes studies reporting epigenetic changes induced by olive oil (OO) through histone modification and DNA methylation mechanisms. Finally, non-coding RNAs are not translated into a protein, but are transcribed from DNA. They participate—in various forms—in the regulation of gene expression. There are different types of non-coding RNAs, but in this paper we focused on studies where the modulation of microRNAs (miRs) by olive oil and its phenolic components was assessed. MiRs, about 18–25 nucleotides in length, were identified for the first time in 2001 by Lagos-Quintana et al. [197]. The function and biogenesis of miRs has been predicted by *lin-4* and *let-7*, which were firstly identified by genetic analyses of *Caenorhabditis elegans* [198,199]. Developmental timing is generated in the cell nucleus as immature particles (pri-miRNA), which are recognized by the nuclear protein DGCR8, associated with the enzyme Drosha to release hairpins from pri-miRNAs and produce the pre-miRNAs. Pre-miRNA hairpins are exported by exportin-5 to the cytoplasm, where the RNase III enzyme Dicer interacts with the 3 end of the hairpin and cleaves the loop joining the 3 and 5 arms. Finally, two strands are generated, one that is incorporated into the RISC complex and another that is degraded. After being processed, miRs act principally as transcriptional repressors of mRNA expression [200,201]. MiRs do not need to be totally complementary to their seed region of mRNAs; therefore, the alteration of a single micro-RNA can change the expression of multiple genes [202]. For this reason, the regulation of miRs through diet or through pharma-nutritional interventions is being proposed as a valuable therapeutic strategy in various diseases, because it would modulate functionally-related pathway genes via epigenetic changes. The literature reports many changes in miR profiles induced by the consumption of different types of OO, namely EVOO. In animal model studies, aged mice were treated with extra-virgin olive oil rich in phenols (6 mg/kg) for six months, and miR modulation in brain tissue was observed; such modulation appears to exert positive regulatory effects on neuronal function [203]. Epigenetic investigations were performed in pregnan<sup>t</sup> Sprague–Dawley rats fed with different oils, i.e., soybean oil (SO), OO, fish oil (FO), linseed oil (LO), or palm oil (PO), from conception to day 12 of gestation and with a standard diet thereafter. MiRs expression was assessed in the liver and in adipose tissue. The results show that maternal consumption of different types of oils influences miR expression and may epigenetically explain the long-term phenotypic changes of the offspring [204]. Regarding human studies (Figure 1), we found two studies in which researchers analyzed the epigenetic changes (through miR assessment) occurring after OO consumption. The interaction of an miR target site SNP with diet and its effects on triglycerides and stroke is one of the many studied outcomes of the PREDIMED trial. In this study, 7187 participants were assigned to three groups: (1) low-fat diet (control); (2) EVOO- or (3) nut-supplemented Mediterranean diet. Researchers found that miR-410 regulated lipoprotein lipase variant rs13702, which is associated with stroke incidence and controlled by diet [205]. Another human research study addressing the effects of supplementations with acute high- and low-phenols EVOO intake on miRs expression was performed on PBMCs of healthy subjects and patients with metabolic syndrome (MS). The result indicated that high-biophenols EVOO intake is able to modify the miR profile; these potentially relevant effects are stronger in healthy subjects [206].

Specific to OO phenolics, some studies analyzed epigenetic changes in miRs produced by HT and/or OLE. Studies in cell cultures with OLE at 200 μM (i.e., non-physiological concentrations) noted that human NPC cell lines and a xenograft mouse model, both irradiated, underwent strongly enhanced radiosensitivity via reduction of the activity of the HIF1 α-miR–519d–PDRG1 pathway, which is essential to radiosensitization [207]. In a study where human ovarian cancer cell lines were used for xenograft assay and were irradiated and treated with 200 μM of OLE, the treatment altered the miR expression profile, specifically; the endogenous expression of miR-299 was repressed by a hypoxia inducible factor and reassured with OLE treatment [208].

To the best of our knowledge, there are no studies that report modulation of miRs by Tyr. Conversely, two papers addressed the actions of HT. In one study, HT modulated the expression of several miRs. In mice supplemented with nutritionally relevant amounts of HT (0.03 g), for eight weeks, changes were found in the expression of miRs in the intestines. The analysis of other tissues revealed consistent HT-induced modulation of only few miRs, e.g., miR-483. In vitro mechanistic studies that used treatment with HT at 10 μM of a human colonic adenocarcinoma cell line (Caco-2), human primary epithelial intestinal cells (InEpCells), and mouse primary organoids confirmed modulation of these miRs. Lastly, one miRNA, miR-193a, was modulated in healthy volunteers supplemented with HT for one week [209]. In a study aimed at elucidating the mechanisms via which OO biophenols modulate miRs, HT, but not OLE (both at 10 μM), induced NRf2 nuclear translocation and reduced miR-146a expression in macrophage RAW 264.7 cells with induced inflammation [210]. Taken together, these studies sugges<sup>t</sup> that both EVOO and its phenolic compounds, together or separately, have effects on the modulation of miRs. In other words, the use of EVOO as principal source of fat modulates our genes through epigenetic changes. Before solid conclusions can be drawn, we would like to underscore that this is a very broad field of research, in which many more studies need to be done. For example, the use of long-term generational research will eventually uncover the true effect of epigenetic changes reported thus far. In addition, future studies will elucidate the possible beneficial effects attributed to the moderate consumption of EVOO in terms of nutrigenomic and epigenetic consequences.



DOA: decarboxymethyl oleuropein aglycone; MedDiet: mediterranean diet; EVOO: extra virgin olive oil. CO: coconut oil; OO: olive oil; SO: sunflower oil; LCO: low corn oil; HCO: high corn-oil; OLE: oleuropein aglycone; *n*-3 LCPUFA: *n*-3 long-chain polyunsaturated fatty acids; PBMCs: peripheral blood mononuclear cells.
