New Mechanisms of Mitochondrial Toxicity: Focus on Antibacterial Agent Cetylpyridinium Chloride Effects on Immune and Barrier Cells

/ABSTRACT CPC is increasingly found at high doses in hygiene, cleaning, pharmaceutical, and food products and is a triclosan replacement but is several-fold more mitotoxic than it and other banned mitotoxicants. Mounting evidence suggests CPC is bioavailable, retained in tissue, toxic to eukaryotes at low doses, and present in humans. Mitochondrial dysfunction/deformation lead to neurological, immunological, and other diseases. We discovered that exposure-relevant, non-cytotoxic CPC doses potently perturb mitochondria (ATP, morphology, oxygen consumption) and cause Ca2+ efflux from mitochondria, but the mechanisms remain unknown. This project will determine the underlying mechanisms and outcomes of CPC mitochondrial toxicity and, critically, will promulgate new general mechanisms of mitotoxicity. Our preliminary data show CPC impedes multiple electron transport chain (ETC) components. CPC also suppresses immune cell signaling via interference with phosphorylation, Ca2+ dynamics, and protein binding to key signaling lipid phosphatidylinositol 4,5- bisphosphate (PIP2). Preliminary data reveal CPC disruption of PIP2 diffusion and cluster properties at the plasma membrane. PIP2 is also found at the outer mitochondrial membrane and its disruption leads to mitochondrial fission--though understanding of PIP2’s mitochondrial biology is in its infancy. We hypothesize that CPC interferes with PIP2, phosphorylation, Ca2+, mitochondrial reactive oxygen species (mtROS), and specific ETC Complexes/associated lipids and enzymes, thus disorganizing mitochondrial shape and function. Using immune (mast and T cells) and barrier (skin, oral, GI) cells, we will assess CPC effects on the ETC using electron flow, qPCR, Western blotting (WB), and measures of mtROS and cardiolipin. ETC disruption outcomes (ATP, glycolysis, lactic acid) will be determined. Kinetics of TCA enzymes in the presence of CPC will be assessed. Super-resolution fluorescence photoactivation localization microscopy will interrogate nanoscale CPC effects on mitochondrial morphology and PIP2 clusters and protein interactions at mitochondria, revealing the fundamental biology of this critical signaling lipid at the mitochondrion. Confocal imaging will provide another perspective, including mitochondrial translocation in the cell. We will assay CPC effects on mitochondrial fusion/fission proteins with qPCR, confocal of subcellular dynamics, and WB, including of phosphorylation status. We will determine the mechanism by which CPC affects the AMPK/mTOR pathway, which leads to mitochondrial fission, by assessing phosphorylation status with WB. We will extend the findings with a proven animal model for mitochondrial research, C. elegans, assessing CPC effects on mitochondrial stress responses, mitochondrial membrane potential, and dopaminergic neurodegeneration. This research will uncover the mechanisms underlying CPC disruption of mitochondrial function in order to fulfill an urgent need by providing insights into CPC effects on environmental human health. It will also define new general modes of mitotoxicity for consideration in toxicity testing workflows. Project Number: 1R15ES037871-01 | Fiscal Year: 2025 | NIH Institute/Center: National Institute of Environmental Health Sciences (NIEHS) | Principal Investigator: Julie Gosse | Institution: UNIVERSITY OF MAINE ORONO, ORONO, ME | Award Amount: $526,441 | Activity Code: R15 | Study Section: Environmental Determinants of Disease Study Section [EDD] View on NIH RePORTER: https://reporter.nih.gov/project-details/11220937