(P22-058-25) The Regulation of Zinc Homeostasis in Senescent Intestinal Cells

Objectives: Aging intestines accumulate cells with senescent characteristics despite the regular regeneration of intestinal epithelium. As the primary site of dietary zinc (Zn) absorption, aged enterocytes have been implicated in impaired Zn absorption. However, the mechanisms underlying this dysfunction remain unclear. This study aimed to investigate dysregulated Zn homeostasis in senescent enterocytes and to elucidate the mechanistic role of Zn in senescence.

Methods: Senescence was induced in C2BBe1 cells through etoposide treatment and irradiation. Senescence induction was validated by assessing senescence-associated markers, including gene expression, protein expression, and β-galactosidase staining (Sa-ß-gal). Zn transporter gene expressions were analyzed using RT-qPCR, and cellular Zn levels were quantified using a colorimetric Zn assay and fluorescent imaging. Zn distribution within the Golgi apparatus was examined using fluorescence microscopy, combining a Golgi-specific marker. Golgi Zn was measured using a genetically encoded Förster Resonance Energy Transfer (FRET) sensor, and analyzed using FIJI software. A membrane-permeable Zn chelator was applied to deplete intracellular Zn levels during senescence establishment. Statistical analyses were conducted using GraphPad Prism 10.

Results: Etoposide administration and irradiation induced senescence, as evidenced by increased (CDKN1A, IL-6) or decreased (LMNB1) expression of senescence-associated genes, elevated P-21, and an increased percentage of Sa-ß-gal-positive cells. Cellular Zn levels were significantly elevated, as determined by both detection methods. Gene expression analysis revealed the upregulation of SLC30A7, a gene encoding a Golgi-associated Zn transporter, across both senescence induction methods. Fluorescent imaging demonstrated intracellular Zn accumulation, including within the Golgi, that was further validated using the FRET sensor. Zn chelation during senescence progression showed a trend toward reduced senescence development, indicated by senescence-associated markers.

Conclusions: Our model exhibits that Zn accumulation, particularly in the Golgi, may contribute to the progression of senescence in intestinal epithelium and Golgi dysfunction.

Funding Sources: This work is partially supported by the USDA’s NIFA program.