Cellular stress, inflammation and barrier damage in gut epithelial cells caused by aspartame

Aspartame has been widely used as a sweetener in foods and beverages since the 1980s. In this study, we aimed to assess the effect of aspartame on gut epithelial cell biology, inflammation, and the epithelial barrier. Aspartame shows cytotoxic effects and epithelial barrier damage as well as proinflammatory cytokine release in gastrointestinal epithelial cells, organoids and organ-on-a-chips at concentrations corresponding to daily used doses in food products. Cellular cytotoxicity was observed in as low as 1.25 mg/ml doses. RNA sequencing analysis revealed that aspartame significantly altered the transcriptome in gut-on-a-chip models, showing upregulation of pathways such as unfolded protein response, activated pro-apoptotic and inflammatory, and downregulation of DNA repair and replication mechanisms pathways. Aspartame exposure upregulated proinflammatory genes, particularly in the TNF signalling pathway, and induced multiple chemokine responses. It activated the NF-?B pathway via oxidative stress, promoting inflammation on NF-?B reporter monocyte cells and causing gut epithelial cell death. Aspartame affected genes involved in tight and adherens junctions, disrupting gut epithelial barrier integrity in a dose-dependent manner in gut-on-a-chip models. Additionally, aspartame suppressed key DNA repair and replication genes involved in double-strand break repair, mismatch repair, and DNA replication. Overall, our findings indicate that at commonly consumed levels, aspartame induces cellular stress, inflammation, and epithelial barrier damage in gastrointestinal epithelial cells. These findings underscore the biological relevance of our results and raise concerns that daily dietary intake of aspartame may pose previously underappreciated risks to gut health. Overall design: A gut-on-a-chip system was used to assess the effect of aspartame on colon epithelial cells. Gut-on-a-chip cultures were established with OrganoPlate® 3-lane 64 plates (Mimetas BV, Leiden, The Netherlands) following the manufacturer's protocols.On day 8 of gut-on-a-chip culture, the chips were exposed to 10mg/ml and 2.5mg/ml aspartame dissolved in culture media for 24 hours. RNA from the gut-on-a-chip culture were harvested at 24 hours of the aspartame exposure.