Iron-mediated epigenetic activation of NRF2 targets

The toxic effects of excess dietary iron within the colonic lumen are well documented, particularly in the context of Inflammatory Bowel Disease (IBD) and Colorectal Cancer (CRC). Proposed mechanisms that underpin iron-associated intestinal disease include: i) the pro-inflammatory and ROS-promoting nature of iron, ii) gene-expression alterations, and iii) intestinal microbial dysbiosis. However, to date no studies have examined the effect of iron on the colonic epigenome. Here we demonstrate that chronic iron exposure of colonocytes leads to significant hypomethylation of the epigenome. Bioinformatic analysis highlights a significant epigenetic effect on NRF2 (nuclear factor erythroid 2-related factor 2) pathway targets (including NAD(P)H Quinone Dehydrogenase 1 [NQO1] and Glutathione peroxidase 2 [GPX2]); this demethylating effect was validated and subsequent gene and protein expression quantified. These epigenetic modifications were not observed upon the diminishment of cellular lipid peroxidation with endogenous glutathione and the subsequent removal of iron. Additionally, the induction of TET1 expression was found post-iron treatment, highlighting the possibility of an oxidative-stress induction of TET1 and subsequent hypomethylation of NRF2 targets. In addition, a strong time dependence on the establishment of iron-orchestrated hypomethylation was found which was concurrent with the increase in the intracellular labile iron pool (LIP) and lipid peroxidation levels. These epigenetic changes were further validated in murine intestinal mucosa in models administered a chronic iron diet, providing evidence for the likelihood of dietary-iron mediated epigenetic alterations in vivo. Furthermore, significant correlations were found between NQO1 and GPX2 demethylation and human intestinal tissue iron-status, thus suggesting that these iron-mediated epigenetic modifications are likely in iron-replete enterocytes. Together, these data describe a novel mechanism by which excess dietary iron is able to alter the intestinal phenotype, which could have implications in iron-mediated intestinal disease and the regulation of ferroptosis.