LOW-DOSE SULFITE EXPOSURE DYSREGULATES EPITHELIAL AMINO ACID METABOLITES AND ENERGY METABOLISM IN PEDIATRIC COLON ORGANOIDS

Abstract Environmental factors contributing to pediatric digestive diseases remain poorly characterized. Sulfites, commonly used as food preservatives, are disproportionately consumed by children, yet current labeling regulations only mandate disclosure above 10 parts per million (ppm). The molecular consequences of low-dose sulfite exposure on intestinal epithelial function are largely unknown. We investigated the impact of sub-threshold sulfite exposure on epithelial metabolism and differentiation using biopsy-derived colon organoids from pediatric patients with active ulcerative colitis (aUC), inactive UC (iUC), and non-IBD controls (n = 3/group). Spheroids were exposed to sodium sulfite either acutely in the undifferentiated state or continuously during differentiation. Short-term exposure (≤200 μM, 16 hrs) in undifferentiated control spheroids dose-dependently increased oxygen consumption. To assess transcriptomic changes during differentiation, spheroids were pre-exposed to 50 μM sodium sulfite (6.4 ppm) for 16 hrs, washed, and differentiated for six days (without sulfite). Single-cell RNA sequencing (using split-pool combinatorial barcoding) and DAVID Gene Ontology analysis of downregulated genes, aUC vs control, in Lgr5-rich stem cell clusters revealed enrichment of amino acid transport and the signal recognition particle (SRP) which are critical for mRNA translation and ER protein maturation. Upon sulfite exposure, functional annotation of downregulated genes of stem cell clusters equally showed significant enrichment of SRP genes. Notably, RN7SL1 and RN7SL2, noncoding RNA components of the SRP, were suppressed in control stem cells. Among all mature epithelial cell types in control, enteroendocrine clusters showed significant transcriptional dysregulation (15 upregulated, 10 downregulated genes), suggesting the impact of one-time sulfite pre-exposure on enteroendocrine cell function. Chronic low-dose exposure (50 μM, 3 days) during differentiation significantly reduced oxygen consumption and ATP production, with iUC colonoids exhibiting heightened vulnerability. Untargeted metabolomics revealed preferential depletion of amino acid metabolites in sulfite-exposed control colonoids. These findings suggest that even low-level sulfite exposure can impair epithelial energy metabolism and differentiation, potentially mimicking metabolic dysfunctions observed in IBD. Ongoing studies will determine whether chronic sulfite exposure exacerbates inflammation and barrier permeability. This research was funded by: 1. 1K99DK136971- NIH/NIDDK 2. Stanford Center for Pediatric IBD and Celiac Disease 3. Stanford Maternal and Child Health Research Institute