To evaluate the significance of the commonly methylated genes ( n = 1187 ) , categorization was implemented . The commonly affected pathways were prominently plasma membrane - associated receptors and components , cytoskeleton and adhesion / junction - related proteins , ion channels / transporters and energy homeostasis ( Figure 3H ) . Considering the distinct physicochemical properties between AgNPs and GO , pathway analysis was further performed for the differentially methylated genes ( Figure 3I , J ) . A multitude of genes were found differentially methylated between AgNPs and GO in both A549 and 293 T cells , including genes responsible for gap junction ( e.g. , LPAR1 and GNAI3 ) [ 24 ] upon AgNPs exposure and genes encoding proteins involved in substance metabolism ( e.g. , PDE5A and GFPT1 ) [ 25 ] upon GO exposure . These differences between AgNPs and GO could be attributed to their distinct physicochemical properties and their different mode of interaction with cells including differential subcellular localization . [ 26][27][28][29 ] For example , AgNP dissolves ions , and Ag ions play a major role in AgNP - induced toxic effects ; [ 26 ] GO have different lateral sizes and surface functional groups and have strong association with biomolecules . [ 27 ] Therefore , we looked into the changes of 5 - mC levels responding to Ag ions and GO with different lateral sizes and surface modifications . Previous studies demonstrated that Ag particles and Ag ions simultaneously existed inside the cells with a release rate of Ag ions around 10 % . [ 28,30 ] Thus , Ag ions at 0.4 µg mL −1 were used to treat cells , and different from Ag particles , Ag ions did not induce a significant change in 5 - mC content in A549 , 293 T , and THP-1 cells ( Figure S14 , Supporting Information ) . In order to answer whether length / size contributes to the influence on DNA methylation induced by ENMs , we deliberately prepared an array of GO with different lateral size , i.e. , S - GO ( smallest ) , I - GO ( intermediate ) , and L - GO ( largest ) , as described in our recent report . [ 28 ] As shown in Figure S18 ( Supporting Information ) , no significant difference could be found among these sized - GO in increasing 5 - mC levels in A549 and 293 T cells . Furthermore , we scrutinized the effect of surface modification on DNA methy lation changes using GO - NH 2 and GO - PEG ( polyethylene glycol coated GO ) , as described in our recent study . [ 31 ] As shown in Figure S19 ( Supporting Information ) , compared with the parental GO and GO - NH 2 , GO - PEG induced a milder increase of 5 - mC levels in A549 and 293 T cells , in agreement with the greater biocompatibility of GO - PEG . [ 31 ] Taken together , these data revealed new insights into the structure - activity relationship for AgNPs and GO : Ag nanoparticles rather than dissolved Ag ions were responsible for the DNA methylation changes ; the surface activity of GO rather than its size played the major role in GO - induced DNA alternations . However , further studies should be considered to identify the physicochemical determinants and nanobio interactions ( e.g. , distinct subcellular localizations ) in inducing different signatures of DNA methylation for diverse ENMs in the future .