Date of Award

Summer 8-12-2017

Level of Access Assigned by Author

Campus-Only Thesis

Degree Name

Doctor of Philosophy (PhD)

Department

Biomedical Sciences

Advisor

Julie A. Gosse

Second Committee Member

Carol Kim

Third Committee Member

Samuel Hess

Additional Committee Members

Roger Sher

Douglas Curie

Abstract

Triclosan (TCS) is an antimicrobial used so ubiquitously that seventy-five percent of the U.S. population is likely exposed to TCS via consumer goods and personal care products. In September 2016, TCS was banned from soap products following the FDA’s risk assessment. However, TCS still remains in other consumer products such as toothpaste, mouthwash, hand sanitizer, and surgical soaps. TCS is readily absorbed into human skin and oral mucosa and has been found in various human tissues and fluids. TCS is known to cause physiological and health problems, including muscle effects in animal models, reproductive defects in human, and decreased infant head circumference. In clinical studies TCS has also been shown to alleviate allergic skin disease, which is caused in part by mast cell over stimulation. Mast cells are ubiquitous immune effector cells that, upon stimulation, release chemical mediators via a process termed degranulation. Proper immune and neurological function requires mast cell activation, but mast cells are also involved in allergies, other forms of atopy, and neurological diseases. The ubiquity of mast cells throughout connective tissues and along epithelial surfaces makes them prime targets for TCS exposure. In this body of work, we have developed new protocols for working with both triclosan and mast cells.

Using these and additional methods, we show that TCS inhibits mast cell degranulation, by lowering cytosolic calcium levels. TCS also inhibits mitochondrial translocation to the plasma membrane via disruption of microtubule polymerization--also contributing to triclosan's inhibition of degranulation.

Upon further investigation into molecular mechanisms underlying triclosan’s inhibition of degranulation, we discovered that TCS is a proton ionophore mitochondrial uncoupler in mast cells (human and rat), mouse fibroblasts, and primary human keratinocytes. In these cells, non-cytotoxic doses of TCS cause the following hallmarks of mitochondrial uncouplers: inhibited ATP production, increased oxygen consumption rate, and decreased mitochondrial membrane potential. Triclosan-methyl, which has a methyl group in place of triclosan's ionizable hydroxyl group, neither inhibits degranulation nor decreases ATP production, indicating that triclosan's effects are due to its proton ionophore structure. TCS also disrupts mitochondrial morphology. This change includes inducing a toroid or "donut" morphology or an increase in mitochondrial fission. The underlying mechanisms include TCS-induced increases in cytosolic ROS production and alterations in mitochondrial and ER calcium levels. Interestingly, we also find that TCS is much more toxic to mitochondria than the banned mitochondrial uncoupler, 2,4-dinitrophenol. Our findings provide a mechanism for TCS disruption of both mast cell degranulation and universal dysfunction of mitochondria. These biochemical effects could help explain epidemiological studies showing adverse effects in humans.

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