## 7 Challenges
Additionally, buccal cells also offer potential advantages to human epigenetic studies, as they represent a better surrogate tissue for brain tissues, with both being ectodermal tissues, and because they can be collected via a non-invasive method (buccal swabs) [156]. Finally, it should be noted that statistical methods to account for cell composition in DNA methylation assays are implemented and available [157]. Seafood is not the only source of exposure to Hg and PAHs. Working with dental amalgam fillings and working or residing among artisanal and small-scale gold mining sites result in elevated exposures to elemental and inorganic Hg [12], which may lead to DNA methylation changes [83; 158]. In the same way, several occupations, such as coke oven manufacturing, chimney sweeping, paving and roofing, entail relevant exposure to PAH mixtures [159; 160], with consequent impacts on the DNA methylation status [86; 161]. Asking participants about their occupation is therefore of fundamental importance in order to address potential confounders of the association between exposure level to MeHg or PAHs via seafood consumption and DNA methylation changes. As regards the influence of genetics on the individual's biological response to MeHg and PAHs exposure, it should be considered that, sometimes, the same genes are involved in the toxicokinetic of and/or susceptibility to different substances. This, obviously, complicates the detection of genes that may be subject to natural selection driven by a specific chemical. This is the case of GSTP1, MT4 and ALAD genes, whose variation can influence the toxicokinetic of Hg (Tables 1 and S1), but also of Cd [162] and Pb [163, 164, 165]. The same is true for NAT2 gene polymorphisms, which influence the toxicokinetics of both Hg [65] and PAHs [69] (Tables 3 and 4). A further level of complexity is related to the fact that concentrations of these chemicals in edible tissues of aquatic organisms are influenced by several factors. Several studies have highlighted the role of trophic level, habitat and size of the organism in determining the level of MeHg in sea animals. In particular, despite discordant evidence [166, 121], MeHg concentration in fishes often increases with trophic level [126, 167], size and age [47], with MeHg uptake being a process of bioaccumulation during the whole life [118], and other studies show that MeHg concentrations are affected also by changes in feeding habits during fish lifespan [132]. The relationship between size and MeHg concentration in marine invertebrates is rather less clear: while some results point to a positive correlation between these two variables in bivalves and crustaceans [124, 168], others show a negative [120, 169] or no significant correlation [124] in the same taxonomic classes, or even in the same species. As regards PAHs, only few studies have tried to assess the influence of biological factors on their accumulation in aquatic organisms [8], and these led to discordant results. Several studies found no significant correlation between PAHs concentrations and fish size or age [170, 171, 172, 173], while [PERSON] and colleagues [28] found a negative correlation between body size and PAH concentrations in the liver and gills of common soles caught in the Po Delta and off Chioggia. Additionally, the sex [170, 173, 174] and the reproductive stage [8] of the organism seem to affect PAHs accumulation and metabolism in fish. MeHg and PAHs concentration in fish also vary with seasons [8, 171, 175, 176, 177, 178] and with the geographic origin of the fished specimen, with some areas of the Mediterranean being more polluted than others. Addressing the above factors through a questionnaire is not easy, if not impossible, which means that a proper estimate of the habitual seafood intake or, more generally, of the eating habits of the sampled individuals, does not always correspond to a precise estimate of their habitual MeHg and/or PAHs intake [179]. To overcome these limitations, the most effective solution is the use of biomarkers of exposition.