A BDE-47/estrone mixture modulates macrophage immune responses and miRNA networks, impairs intestinal barrier integrity in vitro, and alters circulating miRNAs and tight junction expression in vivo

Human exposure to environmental contaminants typically involves complex mixtures rather than single chemicals, complicating health risk assessment. In this study, we investigated the effects of 2,2',4,4'-tetrabromodiphenyl ether (BDE-47) and estrone (E1), individually and in combination (MIX-3), on immune and intestinal epithelial cells. Murine macrophages (RAW 264.7) exposed to BDE-47, E1, or MIX-3 showed no cytotoxicity or oxidative stress at the tested concentrations. However, MIX-3 significantly increased inflammatory gene expression (TNF-α, IL-6, IL-10) and cytokine secretion (TNF-α), suggesting a synergistic immune-activating effect. Intracellular miRNA profiling revealed downregulation of immune-regulatory miRNAs (let-7a-5p, miR-423-5p, let-7c-5p, miR-128-3p), supporting activation of inflammatory and cytokine-related pathways. In Caco-2 cells, MIX-3 exposure reduced tight junction markers (E-cadherin, occludin) and transepithelial electrical resistance, indicating impaired epithelial barrier function, whereas treatment with conditioned medium from MIX-3-exposed macrophages partially restored E-cadherin expression, suggesting a protective role of immune cell-derived soluble factors. To validate these findings in vivo, Wistar rats were orally exposed to the BDE-47/E1 mixture for 30 days. mRNA analysis and histological examination of intestinal tissue highlighted occludin downregulation and mild tissue alterations, consistent with subtle epithelial barrier impairment. Circulating miRNA profiling showed upregulation of inflammation-associated miRNAs (miR-21-5p, miR-150-5p, miR-142-3p, miR-363-3p), linked through bioinformatic analysis to immune dysregulation, intestinal cancer, and neurotoxicity. Overall, these results indicate that low-dose exposure to pollutant mixtures can induce subtle but biologically relevant immune and epithelial changes, emphasizing the importance of mixture-based approaches in environmental risk assessment.