Mechanisms of Antimicrobial Agent Cetylpyridinium Chloride Mitochondrial Toxicity in Rodent and Primary Human Cells: Super-resolution Microscopy Reveals Nanostructural Disruption

Abstract

People are exposed to high concentrations of antibacterial agent cetylpyridinium chloride (CPC) via personal care and food products, despite little information regarding CPC effects on eukaryotes. CPC is used as an antibacterial agent via a detergent mechanism when above ~600-900 μM. While three previous studies suggested CPC mitochondrial toxicity, this phenomenon is not well-studied. Here, we show that low-micromolar CPC inhibits mitochondrial ATP production in primary human keratinocytes, mouse NIH-3T3 fibroblasts, and rat RBL-2H3 immune mast cells, in galactose media, which causes cells to produce ATP via mitochondria. ATP inhibition via CPC (EC50 1.7 μM) is nearly as potent as that caused by canonical mitotoxicant CCCP (EC50 1.2 μM). CPC inhibition of oxygen consumption rate (OCR) tracks with that of ATP: OCR is halved due to 1.75 μM CPC in RBL-2H3 cells and 1.25 μM in primary human keratinocytes. Here we demonstrate that CPC is more potent as a mitotoxicant than as an immune mast cell signaling inhibitor, an effect published previously. Mitochondrial [Ca2+] changes can cause mitochondrial dysfunction. Here we show, using a novel plate reader assay with reporter CEPIA2mt, that CPC causes mitochondrial Ca2+ efflux from mast cells via an ATP-inhibition mechanism. Using super-resolution microscopy (fluorescence photoactivation localization) in live cells, we have discovered that CPC causes mitochondrial nanostructural defects in fibroblasts, including the formation of spherical structures with donut-like cross section, as quantified by novel Fourier transform analysis. This work reveals CPC as a mitotoxicant despite widespread use, highlighting the importance of further research into its toxicological safety.

Pharmaceutical agent cetylpyridinium chloride inhibits immune mast cell function by interfering with calcium mobilization

“…However, the CPC effects on mast cells described in this article are not caused by the detergent action of CPC because experiments in this study were conducted with CPC doses (0.1-10 µM) far lower than the CMC. Furthermore, CPC doses used in this study are not cytotoxic to various cell types, as determined by trypan blue exclusion (Raut et al, 2022), lactate dehydrogenase release (Raut et al, 2022), ToxGlo (Weller et al, 2022), and plasma membrane potential (Figure 5B) assays. Thus, the CPC exposures used in this study are affecting signal transduction, not cell viability.…”

“…These biochemical effects could be potentially involved in the observed MC inhibition of CPC. However, CPC likely does not hamper MC function (Figure 1) via mitochondrial dysfunction because these degranulation assays are carried out in glucose, in the presence of which CPC does not inhibit mitochondrial ATP production (Weller et al, 2022). Estrogens are known to enhance MC degranulation .…”

“…Based upon this mouse pharmacokinetic study and levels of CPC in chicken for consumption, we estimated that a typical American may have a blood CPC concentration ranging from ∼0.1 μM -0.3 μM due to chicken consumption alone (Weller et al, 2022). CPC administered at sub-µM levels to zebrafish via swim water led to behavioral changes and altered brain neurotransmitter levels (Dong et al, 2022).…”