Ventura, Célia2026-03-092026-03-092025-05-07http://hdl.handle.net/10400.18/11263Titanium dioxide nanoparticles (TiO2NPs) are nanomaterials with relevant properties for several applications in biomedicine and industry, where they are primarily used as a white pigment to improve the opacity, brightness, and whiteness of several products, such as paint, food, cosmetics, and pharmaceutics. Nevertheless, there are still concerns about its effects on human health, particularly through ingestion of the food-grade TiO2NPs, which caused its ban from all food products in the European Union. Moreover, in recent years, there has been a great interest in the role of epigenetics in the biological response to chemical exposure, including the study of DNA methylation. This study aimed at identifying changes in DNA methylation of Caco-2 intestinal cells caused by exposure to anatase (NM-102), rutile (NM-103) and anatase/rutile (NM-105) TiO2NPs, either after a simulated in vitro digestion or the undigested counterparts. Using Reduced Representative Bissulfite Sequencing, significant differential methylation of 209 and 319 genomic regions in cells exposed to undigested and digested NMs was identified, respectively. These included 48, 41 and 55 differentially methylated genes for undigested NM-102, NM-103 and NM-105, and 71, 65 and 55 for the digested counterparts. Reactome pathways and Gene Ontology (GO) analyses showed that both undigested and digested TiO2NPs have a functional impact on intestinal cells, often cancer-related, and that digestion seems to reduce this impact. Different TiO2NPs caused enrichment of different pathways and GO terms, probably due to their specific physicochemical properties. A trend towards CpG hypermethylation was observed upon NM-105 exposure, unlike the other TiO2NPs. This study highlights the importance of DNA methylation analysis in assessing the adverse effects of nanomaterials on human cells.engTitanium Dioxide NanoparticlesTiO2NPsDNA MethylationGenotoxicidade Ambientallearning object