Background: Cellulose nanomaterials (CNMs) are emerging materials under development for various applications in biomedicine and the food industry. Consequently, their potential human health effects warrant evaluation, particularly after oral exposure. Previous studies on two innovative CNMs, CNF-TEMPO and CMF-ENZ, indicated that genotoxicity could not be ruled out, as DNA damage was observed in Caco-2 cells. Whether these CNMs may affect gene expression regulation and induce downstream effects not assessed through genotoxicity testing, is still an open question. This work presents a new approach methodology coupling in vitro simulated digestion with epigenomics, to investigate changes in DNA methylation upon exposure of intestinal cells to these CNMs.

Results: DNA methylation patterns differed upon cell exposure to undigested or digested samples, showing a trend towards hypermethylation for undigested and hypomethylation for digested samples. Using Reduced Representation Bisulfite Sequencing, significant differentially methylated regions (DMR) and genes were identified in undigested and digested CNMs-exposed cells. Pathway analysis revealed that CNMs influenced multiple signal transduction pathways. CMF-ENZ and CNF-TEMPO impacted glycosaminoglycan metabolism and glycosylation pathways, possibly involved in cell proliferation. CMF-ENZ affected a DNA repair pathway, while CNF-TEMPO impacted pathways associated with glucose metabolism, independently of the digestion process. Both CNMs seemed to affect pathways implicated in overall cytoskeletal and extracellular matrix dynamics. For digested CNMs, particularly digested CNF-TEMPO, this impact suggests a possible deleterious disturbance given the involvement of ERBB2/PIK3 signaling.

Conclusions: This study shows that CNMs may have a biological effect on Caco-2 cells, affecting DNA methylation, and highlights the importance of epigenetic analysis in pointing out the way for unraveling the mechanisms underlying their toxicity.