114 | Mechanisms of antimony-induced intestinal injury under chronic low-dose exposure: evidence from Caco‑2 cells and protective role of cyanidin-3-o-glucoside

Federica Lina Salamone^1|2, Maria Sofia Molonia¹, Santi Trischitta¹, Antonella Saija¹, Francesco Cimino¹, Antonio Speciale¹ | ¹Department of Chemical, Biological, Pharmaceutical, and Environmental Sciences, University of Messina, Messina, Italy; ²Imbesi Foundation, Messina, Italy.

The global rise in inflammatory bowel diseases has been closely associated with industrialization and growing exposure to environmental contaminants, including heavy metals. However, the impact of chronic, low‑dose exposure to emerging contaminants such as antimony (Sb) on intestinal epithelial homeostasis remains insufficiently defined. Sb is a metalloid widely used as a catalyst in polyethylene terephthalate (PET) manufacturing. It is well documented that Sb can leach from plastic packaging into food and beverages, particularly under thermal or mechanical stresses, raising concerns regarding the health consequences of chronic lowlevel human exposure. At the same time, growing attention has been focused on nutraceutical compounds with protective properties. Cyanidin-3-O-glucoside (C3G), a natural flavonoid abundant in Mediterranean diet, is recognized for its multiple beneficial effects on physiological processes. This study investigates the effects of chronic exposure to low concentrations of Sb(III) on human intestinal epithelium and evaluates the potential protective role of C3G. To mimic chronic dietary exposure in a differentiated intestinal epithelium, Caco‑2 intestinal epithelial cells were exposed throughout the whole cell differentiation period to Sb(III), with or without C3G or tauroursodeoxycholic acid (TUDCA), a specific inhibitor of endoplasmic reticulum (ER) stress. Continuous Sb(III) exposure markedly disrupted intestinal epithelial homeostasis by activating inflammatory pathways, as evidenced by upregulated NF‑κB signaling and increased expression of pro-inflammatory mediators (IL-6, IL-8, and COX-2), while simultaneously promoting apoptotic cell death via modulation of Bcl-2 family proteins (Bax, Bcl-2) and activation of Caspase-3. A coherent mechanistic sequence emerged, in which Sb‑induced oxidative stress triggered ER stress activation, evidenced by increased reactive oxygen species production and by activation of key unfolded protein response (UPR) markers (XBP-1, p-eIF2α, ATF4, GRP78, CHOP), ultimately leading to inflammatory and apoptotic outcomes. Pharmacological inhibition of ER stress by TUDCA significantly alleviated Sb-induced inflammation and apoptosis, confirming ER stress as a pivotal driver of Sb-induced intestinal epithelial dysfunction. Notably, co-treatment with C3G effectively counteracted Sb-induced cellular damage, reducing inflammatory responses, apoptotic processes, oxidative imbalance, and ER stress activation. C3G exerted a multi‑target protective action, mitigating both upstream oxidative events and downstream ER‑stress‑mediated outcomes, thereby preserving epithelial integrity and functionality, highlighting its relevance as a natural protective molecule. Overall, these findings show that even low‑dose, chronic Sb(III) exposure can compromise intestinal barrier stability, a critical determinant of gut health. Moreover, this study identifies C3G as a promising nutraceutical strategy to mitigate Sb-induced intestinal epithelial damage, supporting its potential application in preventing adverse health effects linked to environmental metal contamination in the food chain.