Is the Ubiquitous Antibacterial Agent Triclosan an Uncoupler of Mammalian Mitochondria

Home , Rotenone, Triclocarban, Triclosan

The University of Maine DigitalCommons@UMaine

Honors College

Spring 2015

Hina Hashmi University of Maine - Main

Follow this and additional works at: https://digitalcommons.library.umaine.edu/honors

Part of the Microbiology Commons

Recommended Citation Hashmi, Hina, "Is the Ubiquitous Antibacterial Agent Triclosan an Uncoupler of Mammalian Mitochondria" (2015). Honors College. 211. https://digitalcommons.library.umaine.edu/honors/211

This Honors Thesis is brought to you for free and open access by DigitalCommons@UMaine. It has been accepted for inclusion in Honors College by an authorized administrator of DigitalCommons@UMaine. For more information, please contact [email protected].

IS THE UBIQUITOUS ANTIBACTERIAL AGENT TRICLOSAN AN UNCOUPLER OF MAMMALIAN MITOCHONDRIA?

Hina N. Hashmi

A Thesis Submitted in Partial Fulfillment of the Requirements for a Degree with Honors (Microbiology)

The Honors College University of Maine May 2015

Advisory Committee: Julie A. Gosse, PhD, Associate Professor, Molecular and Biomedical Sciences, Advisor Robert Gundersen, PhD, Chair and Associate Professor, Molecular and Biomedical Sciences Melissa Ladenheim, PhD, Preceptor and Coordinator of Advancement, Honors College Rebecca Van Beneden, PhD, Professor of Biochemistry and Marine Sciences, Associate Director, School of Marine Sciences Steven Mahlen, PhD, D (ABMM) Director, Clinical and Molecular Microbiology, Affiliated Laboratory, Inc., EMMC

Abstract Triclosan (TCS), an antibacterial agent widely found in household and clinical products, is readily absorbed into human skin, but TCS effects on mammalian cells are largely unknown. TCS has been found to alleviate symptoms of human eczema, via an unknown mechanism. Mast cells are ubiquitous, key players in allergy, infectious disease, carcinogenesis, autism, and many other diseases and physiological functions.

One important function of mast cells is release of pro-inflammatory mediators from intracellular granules (degranulation) upon challenge with antigen. Non-cytotoxic doses of TCS inhibit several functions of both human (HMC-1.2) and rat (RBL-2H3) mast cells, including degranulation. Previous work in the Gosse laboratory has shown that

TCS disrupts ATP production without cytotoxicity in RBL-2H3 and HMC-1.2 mast cells in glucose-free, galactose-containing media. In the same glucose-free conditions, 15 µM

TCS dampens RBL-2H3 degranulation by 40%. Thus, TCS affects degranulation and

ATP production in mast cells. TCS-methyl, unlike TCS, was shown to inhibit neither of these processes, suggesting that TCS disrupts cellular energy production in part due to its ionizable proton. Well studied mitochondrial uncouplers [e.g., carbonyl cyanide 3- chlorophenylhydrazone (CCCP)] have been shown to disrupt mast cell function.

Oxygen consumption rate (OCR) is a parameter that has been used to study mitochondrial function, since most cellular oxygen consumption is via mitochondria.

Triclosan at 10 µM was shown to increase the cellular oxygen consumption rate in RBL-

2H3 cells, demonstrating a similar effect as 1 µM CCCP. Taken together, our data indicate that TCS is a mitochondrial uncoupler which disrupts mast cell signaling. This mechanism could underlie TCS toxicity in numerous mammalian cell types.

Dedicated to Samuel and his mother in Tanzania.

iii

Acknowledgements

The completion of this thesis would not have been possible without the support of many who in one way or another contributed to the depths of this study. I would like to express immeasurable appreciation and my deepest gratitude to my thesis advisor, Dr.

Julie A. Gosse, for her guidance, patience, and confidence in my abilities while growing as a scientist. I would also like to extend my gratitude to Lisa Weatherly for her direct contributions to the work presented in this thesis, for teaching me to be persistent in science (especially while troubleshooting), and for her insights during the writing process. I would like to thank Juyoung Shim for teaching me valuable lab skills, for her guidance, and for her motherly advice.

A special word of gratitude is due to all past and present members of the Gosse

Lab. Thank you to Rachel Kennedy, Andrew Abovian, and Alejandro Velez for training me through the ins and outs of lab work, and also for being great mentors. I am grateful to Maxwell Dorman and Erik Gerson for their commitment to research in the lab.

Finally, I would like to acknowledge the support of my family and friends for their understanding and encouragement throughout my educational endeavors. Thank you all for being a part of this journey.

Table of Contents

1 Introduction ...... 1

1.1 Triclosan ...... 1

1.1.1 Triclosan Methyl ...... 3

1.2 Triclosan and Human Health...... 4

1.3 Mast Cells ...... 7

1.3.1 RBL-2H3 and HMC-1.2 Cell Lines ...... 9

1.4 Mast Cell Degranulation ...... 10

1.5 Uncoupling of Oxidative Phosphorylation ...... 14

1.5.1 Effects of Known Mitochondrial Uncouplers ...... 15

1.6 Previous Studies ...... 17

2 Materials and Methods ...... 20

2.1 Cell Culture ...... 20

2.2 Reagents and Buffers ...... 20

2.2.1 TCS media preparation ...... 20

2.2.2 CCCP stock preparation ...... 21

2.2.3 Phenol-red free media ...... 21

2.2.4 Tyrodes Buffer ...... 22

2.3 Mitochondrial Membrane Potential Assays ...... 23

2.4 Oxygen Consumption Rate Assays ...... 24

2.5 Glucose Oxidase and Antimycin A Controls ...... 28

2.6 Data analyses ...... 28

3 Results ...... 29

3.1 TCS Interference in Measuring Mitochondrial Membrane Potential ...... 29

3.2 Optimization and Establishment of Oxygen Consumption Rate Assays ...... 31

3.3 TCS Effect on Cellular Oxygen Consumption ...... 40

3.4 Validation that the Increase in OCR is not due to Toxicant Interaction with

Phosphorescent Oxygen Probe ...... 43

4 Discussion ...... 46

4.1 Discussion of Mitochondrial Membrane Potential Assay ...... 46

4.2 Discussion of Oxygen Consumption Rate Assays ...... 48

5 References ...... 50

Author’s Biography ...... 60

List of Figures

Figure 1: Structures of triclosan and methyl-triclosan ...... 4

Figure 2: Schematic of degranulation pathway in mast cells...... 13

Figure 3: Cytotoxicity and ATP production of RBL-2H3 and HMC-1.2 mast cells exposed to toxicant in glucose or glucose-free media...... 18

Figure 4: Instrument Signal Optimization ...... 27

Figure 5: TCS Interference with Mito-ID Membrane Potential Dye ...... 31

Figure 6: Optimization of Oxygen Consumption Rate Assay Protocol ...... 36

Figure 7: RBL Cell Density Optimization ...... 37

Figure 8: Fluorescence controls to identify source of high background ...... 38

Figure 9: Raw fluorescence of various oil overlays...... 39

Figure 10: Representative linear data of OCR in RBL cells ...... 41

Figure 11: Slope of OCR effects due to toxicant exposure ...... 42

Figure 12: OCR effects in RBL cells in the presence of ± Antimycin A (AA) and ± TCS

...... 44

Figure 13: OCR Measured in Oxygen Depleted, No-Cell Environment ...... 45

vii

1 Introduction

1.1 Triclosan

Triclosan [5-chloro-2-(2,4-dichlorophenoxy) phenol] has been used for decades as an antibacterial and antifungal agent1. Originally developed as a pesticide in Switzerland, triclosan (TCS) was used primarily as an antiseptic agent in surgical scrubs in the 1970’s and 1980’s2. Since then, TCS has been commonly found in household and clinical products such as toothpastes, toys, deodorants, and antiseptic soaps at concentrations around 10 mM3,4. Many consumer products containing TCS are disposed into residential drains, leading to detectable levels in terrestrial and aquatic environments, in wild animals, and in humans. According to a 1999-2000 study done in the United States, it was found that 75.7% of all commercially available liquid hand soaps and 26.4% of bar soaps contain TCS5. TCS is readily absorbed into human skin6-9. The widespread use of

TCS has significance at both an environmental and organismal or cellular level. Though

TCS exposure is widespread, mammalian toxicology and pharmacology remain largely unknown.

Triclosan works as an effective antimicrobial by blocking fatty-acid biosynthesis, permeabilizing the bacterial envelope10. Studies examining lipid synthesis in E. coli have shown that TCS mimics the natural substrate of enoyl-acyl carrier protein reductase

(FabI)11. TCS is a chlorinated, aromatic chemical with an ortho positioned hydroxyl group on one of its phenol rings (Figure 1A). Within the active site of FabI, the phenolic hydroxyl group of TCS binds to a hydroxyl group of the nicotinamide ring of the NAD+ cofactor and also to the phenolic oxygen of tyrosine 156, forming a noncovalent, bi-

substrate FabI-NAD+-triclosan complex1,10. Effectiveness of TCS as a surgical scrub ingredient in clinical healthcare settings has been proven1,12.

Due to its widespread use, there has been concern that resistance may develop to

TCS, compromising its usefulness10,13. Recent studies have shown that the ability of organisms such as E. coli to acquire genetic mutations in the fabI gene or the presence of multidrug efflux pumps can bring rise to resistant organisms. In E. coli, a missense mutation in the fabI gene to fabI[g93v] interferes with TCS’s ability to form a stable

FabI-NAD+-triclosan complex10. TCS has been used to manage infectious disease outbreaks in clinical settings such as controlling a methicillin-resistant Staphylococcus aureus (MRSA) outbreak in a neonatal nursery14, but has also been the cause of hospital outbreaks when bacteria persisted in a TCS-containing soap dispenser15. In a 2007

Pseudomonas aeruginosa outbreak in an Italian hematology unit, a P. aeruginosa strain was isolated from a heavily contaminated TCS soap dispenser. P. aeruginosa is intrinsically resistant to TCS due to various resistance mechanisms16. In the hand soap outbreak, TCS exposure doubled the minimum inhibitory concentration (MIC) to antibiotics such as ciprofloxacin, levofloxacin, tetracycline, and chloramphenicol13,15. In addition, it has been demonstrated that TCS exposure to P. aeruginosa can efficiently select for multidrug resistant derivatives, including resistance to antipseudomonas drugs17. Interestingly, TCS derivatives are being explored for use in treating

Mycobacterium tuberculosis18. Although a link between TCS resistance and antibiotic resistance has been consistently demonstrated in clinical and laboratory settings, research conducted at the population level has contrastingly shown little evidence of cross- resistance to antibiotics associated with consumer use of TCS-containing products9.

U.S. regulatory agencies such as the Food and Drug Administration (FDA) and the Environmental Protection Agency (EPA) have debated issuing a ruling on the regulation of TCS use in consumer products, while various other nations have declared

TCS an environmental toxicant. TCS’s stability at temperatures up to 200°C and its high solubility in organic solvents contribute to its detection in the environment, in food, and in humans2,19-21. TCS has been studied widely with respect to its effects in the environment, such as ecological toxicity in aquatic and terrestrial environments22, but effects of TCS in mammals are not well known.

TCS is an ionizable compound and has a pKa of approximately 7.9. At greater pH, TCS is ionized and exists in its anionic (phenolate) form. In its anionic, phenolate form, TCS is susceptible to photolysis, resulting in the potential production of dioxins when exposed to excessive heat or prolonged sunlight in the environment23.

Additionally, it is thought that only the neutral phenolic form of TCS is toxic to aquatic organisms 24 as ionized, charged molecules are less likely to cross hydrophobic lipid membranes. TCS likely crosses plasma membranes hydrophobically.

1.1.1 Triclosan Methyl

Triclosan extensively contaminates wastewater at concentrations around 3.45 nM to 34.5 nM25,26, and about 90 % of TCS is removed via biodegradation25,27.

Approximately five percent of TCS is biomethylated into triclosan-methyl (2,4,4′- trichloro-2′-methoxy-diphenylether) under aerobic conditions27,28, retaining the same structure as TCS with the exception of a methyl group in place of TCS’s ionizable hydrogen. The chemical structures of triclosan and triclosan-methyl (mTCS) are shown

in Figure 1 for comparison. Triclosan-methyl is known to bioaccumulate more readily than TCS, has greater lipophilicity, and has less sensitivity toward photodegradation in the environment29. Because the hydroxyl group of TCS is largely responsible for its antimicrobial function, mTCS, with a methyl group in place of a hydroxyl group, loses the antibacterial properties of its parent compound. We hypothesize that triclosan’s ability to disrupt mast cell function is due, in part, to its ionizable proton. Based on its structural properties, we suggest that mTCS can be used as a control to test this hypothesis.

A) B)

Figure 1: Structures of triclosan and methyl-triclosan

Triclosan (5-chloro-2-(2,4-dichlorophenoxy) phenol) structure (A) and TCS-methyl (B) Source: PubChem

1.2 Triclosan and Human Health

An increasing number of studies report that triclosan affects many specific biological functions in various species, including humans. TCS has low water solubility

(up to 40 µM30 and unless at alkaline pH31) and is known to be readily absorbed in humans by the gastrointestinal tract8. Additionally, due to its lipophilic properties, TCS has been seen to accumulate in adipose tissue and also in breast milk, amniotic fluid, blood, and urine32. Testing urine is a useful biomonitoring tool for assessing information

on baseline levels of TCS in human subjects. A single oral dose of TCS can be detected in human urine samples for up to 8 days8. In the 2003-2004 National Health and

Nutrition Examination Survey (NHANES), it was reported that 75% of urine samples contained TCS, suggesting that hundreds of millions of U.S. citizens are exposed to

TCS33.

In clinical studies, TCS has been shown to suppress human skin atopic disease34 through an unknown mechanism35. Antiseptics, such as triclosan, are an alternative to topical antibiotics in patients suffering from atopic dermatitis36. One randomized, double-blind study examining the efficacy of 1% TCS-containing emollient in 60 patients with atopic dermatitis reported reduced severity of the skin condition after 14 days34.

Improvement in severity was followed by reduction in topical steroid application by treated patients, as well as reduction in Staphylococcus aureus colonization.

Studies have also evaluated the biological activities of TCS in mammals and its potential adverse effects37. TCS has cytotoxic effects on breast cancer cells38.

Additionally, upon absorption, TCS is rapidly distributed throughout the human body and disrupts endocrine function39, initiates anti-inflammatory responses 40,41, and affects the function of natural killer (NK) cells of the immune system42. Various studies report chemicals in the environment which mimic natural estrogen and affect ER function in cells. Such chemicals disrupt normal endocrine function and raise reproductive health concerns. Studies have proposed that androgenic endocrine disrupting chemicals (EDCs) may reduce sperm production, alter gonad development, and contribute to neurological diseases in males43-45. In vitro studies in human recombinant cells and in vivo studies in aquatic species have suggested that TCS has endocrine disrupting properties and may be

weakly androgenic46. In rats, TCS has been shown to disrupt thyroid hormone homeostasis47 and also inhibit enzymatic activity of sulfotransferase and a glucoronosyltransferase in human liver fractions48.

TCS has been shown to modulate anti-inflammatory effects on various immune cell types, including monocytes, lymphocytes, and neutrophils49. In vitro studies have shown that TCS inhibits cyclo-oxygenase and lipoxygenase pathways in human gingival fibroblasts, decreasing TLR-induced secretion of inflammatory factors such as prostaglandin E2, leukotriene, and interferon-γ49,50. While TCS has been shown to modulate inflammation both in vitro and in vivo, little has been elucidated about its mechanisms of action. A study by Udoji et al has indicated that TCS decreases the target-cell destroying (lytic) function of NK cells, similar to carbamate pesticides51 and brominated flame-retardants52. Human NK cells are a first line of defense protecting against viral infections and the development of cancers53. TCS (10 μM) was shown to inhibit more than 90% of NK function within an hour of exposure and increased upon length of exposure54.

Previous studies support that the pleiotropic effects of TCS indicate that various molecular mechanisms of action are responsible for the diverse biological effects seen in humans. In eukaryotes, TCS effects have been linked to membrane interaction55.

Membranotropic effects which impair metabolic function in isolated rat liver mitochondria exposed to TCS have been examined56. In this thesis, we will explore a potential mechanism of action for TCS disruption of metabolic function which may underlie TCS effects on various biological processes.

1.3 Mast Cells

The Gosse Laboratory has discovered that TCS inhibits mast cell function in vitro57,58. Mast cells are highly granulated immune effector cells that play a role in Type

I hypersensitivity and modulate allergic immune response by releasing pro-inflammatory mediators from intracellular granules. Mature mast cells are found in most body tissues and are key players in not only allergy, but also infectious disease, carcinogenesis, and even a host of neurological functions and diseases such as autism53,59,60. There is recent, widespread evidence that mast cells also modulate behavior61. Because mast cells are most abundant at host-environment interfaces such as mucosal tissues and the skin, they are likely exposed to TCS via use of TCS-containing consumer products. The ubiquitous presence of mast cells in mammals makes them an appropriate model for examining TCS exposure and its effects.

Mast cells are hematopoietic cells that develop from CD34+ /CD117+/CD13+ pluripotent precursor cells, originating in the bone marrow in humans 62. In rodents, these progenitor cells express Thy-1lo c-kithi. Similar to macrophages, mast cells circulate in the bloodstream before they migrate to the peripheral connective or mucosal tissues where they complete their development. Following stem cell factor (SCF)- induced KIT dimerization and auto-phosphorylation, KIT is activated, allowing for mast cell maturation. Substantially reduced mast cell numbers have been shown in mice defective of KIT catalytic activity. SCF regulates mast cell survival and elicits cell-cell or cell-substrate adhesion, and also facilitates proliferation, differentiation and development of mast cells63.

Mast cells are a key component of innate and adaptive immunity. Due to their anatomical locations, mast cells serve as a first line of defense against pathogens such as bacteria, viruses, and parasites. The hallmark characteristic of mast cell morphology is the presence of electron-dense, cytoplasmic granules, the basis of their discovery in the late nineteenth century. The secretory granules of mast cells are composed of preformed chemical mediators including histamine, tryptase, serotonin, and chymase64. Among the bioactive amines present in mast cell granules, histamine is the most clinically significant65. In addition to preformed constituents, upon activation, de novo synthesis of lipid mediators such as leukotrienes and prostaglandins, cytokines, and chemokines are induced66. These mediators influence a variety of cellular responses, including induction of inflammatory response, angiogenesis, tumorigenesis and cellular hyperplasia. Though granule composition may seem homogenous, there is widespread evidence that granule morphology and contents are heterogeneous, as in a subdivision of granules seen in bone marrow-derived mast cells of mice67. Though mast cell degranulation most notably occurs in response to IgE receptor cross-linking, degranulation can occur in response to complement activation, toxins, and neuropeptides. The release of particular mediators from intracellular granules is dependent upon the tissue microenvironment68, and many of the physiological functions of mast cells are associated with the bioactivity of the granule components.

Similar to other leukocytes, mast cells detect their external environment by virtue of a multiplicity of cell-surface receptors, including Fcε, Fcγ, TLRs, major histocompatibility complexes (MHC) classes I and II, KIT, and various interleukins69.

Allergen-induced mast cell signaling in Type I allergic reactions occurs through

aggregation of the membrane embedded FcεRI receptor via antigen cross-linking of IgE to FcεRI on the mast cell and basophil cell surface. The extensively studied rat basophilic leukemia (RBL-2H3) mast cell model, has been used to examine IgE mediated degranulation as well as degranulation by methods which mobilize calcium53,70.

1.3.1 RBL-2H3 and HMC-1.2 Cell Lines

Functionally homologous to human mast cells, rat basophilic leukemia cells, clone 2H3 (RBL-2H3)71,72 have the core functional signaling machinery of mature human mast cells73,74 and have been used to study mast cell signaling for decades. RBL mast cells have been used as a biosensor for screening chemicals for environmental toxicity75.

The RBL-2H3 cell line is a continuous cell line that was cloned from isolated rat leukemia cells treated with β-chlorethylamine, a chemical carcinogen. RBL-2H3 cells have been used extensively for the study of IgE- FcεRI interactions and degranulation signaling pathways76 which are similar to the process seen in human basophils73,74. The

FcεRI receptor can selectively bind murine IgE antibody and can be stimulated by allergen cross-linking to induce the release of granules.

A substantial number of monoclonal cells can be obtained via basic cell culture technique, therefore, RBL-2H3 cells are a useful model for in vitro work and will be utilized in this study. RBL-2H3 cells are a reliable model for the study of IgE-mediated degranulation of secretory cells containing chemical mediators such as histamine, β- hexosaminidase, and serotonin. To increasing concentrations of DNP-BSA antigen,

RBL-2H3 cells exhibit a dose response curve77. This phenomenon in mast cells has also been previously reported in basophils.

The Gosse lab has shown that non-cytotoxic, low micromolar doses of TCS inhibit degranulation57 and suppress ATP production in RBL cells as well as in human mast cells (Weatherly, Shim, Hashmi, Kennedy, Hess and Gosse, submitted). HMC-1 human mast cells, which lack functional FcεRI receptors 78 are immature mast cells and are poorly granulated 79. The human mast cell-1.2 (HMC-1.2) model can be activated by calcium ionophore80. A decrease in antigen (Ag) stimulated degranulation has been seen in RBL-2H3 cells exposed to carbonyl cyanide 3-chlorophenylhydrazone (CCCP), a potent mitochondrial uncoupler81. In this study, we will further examine another key test for mitochondrial uncoupling, namely, identifying a change in oxygen consumption, in the RBL-2H3 cell model as it is exposed to TCS.

1.4 Mast Cell Degranulation

As previously mentioned, an important physiological function of mast cells is the modulation of the allergic immune response by releasing pro-inflammatory mediators from their intracellular granules (degranulation) upon challenge with antigen or allergen, such as pollen, in sensitized individuals82. When mast cells degranulate, all granules undergo exocytosis. This process is physiologically important for the release of intracellular mediators such as histamine at correct sites in tissue. Novel imaging techniques using internal reflection microscopy have been used to analyze recruitment of granules, docking at the plasma membrane, and exocytosis83. The well defined stages within the exocytotic pathway include ATP-dependent priming steps, movement of

vesicles to the subplasmalemmal membrane, docking at release sites, triggered membrane fusion, and release of granule contents.

RBL-2H3 mast cells can be activated by aggregation of the FcεRI receptor by IgE bound to multivalent antigen or by methods which mobilize calcium53. FcεRI is a tetrameric receptor consisting of an α chain and a membrane-tetraspanning β chain 84.

IgE binds the α subunit of the FcεRI receptor, causing antigen cross-linking which prompts the phosphorylation by Lyn of tyrosine residues of the β and γ subunits of the receptor. Phosphorylated sites on the γ subunit recruit the docking of spleen tyrosine kinase (Syk)85. The binding of Syk to the γ subunit induces a conformational change in

Syk and resultant autophosphorylation and liberation from the receptor. Syk catalyzes a series of phosphorylation steps to activate proteins such as the linker for activation of T- cells (LAT) and PI3K86. PLCγ1is recruited to the membrane by LAT where it is phosphorylated by Syk. PLCγ1 catalyzes the hydrolysis of phosphatidylinositol 4,5- bisphosphate (PIP2) to inositol-1,4,5-trisphosphate (IP3) and diacylglycerol (DAG)87.

IP3 initiates a biphasic increase in intracellular calcium, and DAG activates protein kinase C (PKC). IP3 binds its receptor on the endoplasmic reticulum (ER), causing calcium to be released from internal ER stores. Calcium depletion from ER stores prompts a conformational change in STIM1 to allow for interaction with calcium release activated calcium (CRAC) channel/Orai1 in the plasma membrane, resulting in a calcium influx88. Sarco/endoplasmic calcium ATPase (SERCA) pumps permit the reuptake of this calcium to replenish ER stores and sustain cytosolic increase in Ca2+, the essential signal for mast cell activation89. The dynamics and spatial configuration of the

intracellular calcium signal is shaped by the mitochondria which possess a high capacity for calcium buffering as well as specialized mechanisms for Ca2+ uptake and efflux90.

Phosphorylation of protein myosin by PKC allows for cytoskeleton rearrangement to facilitate granule mobilization essential for degranulation. Intracellular calcium and

PKC activate PLD in two isoforms in RBL cells: PLD1, which is involved in granule translocation, and PLD2, which is involved in energy dependent step of membrane fusion of granules where they dock with SNARE proteins91. These signaling processes induce degranulation, or the release of various mediators such as histamine. A schematic of the degranulation pathway is provided in Figure 2.

For the purposes of this study, it is important to note that these processes are energy (ATP) dependent83, and degranulation has been shown to be inhibited in RBL57,58 and HMC mast cells (Weatherly, Shim, Hashmi, Kennedy, Hess and Gosse, submitted) previously.

PLD1

SERCA

PLD2

Figure 2: Schematic of degranulation pathway in mast cells. Figure adapted from Rachel Kennedy and Jonathon Pelletier.

1.5 Uncoupling of Oxidative Phosphorylation

Mitochondria play a crucial role in an array of biological phenomena, including metabolism, bioenergetics, and cancer92. Mitochondrial uncouplers disrupt the proton gradient across the inner mitochondrial membrane, thus disrupting the coupling between electron transport and oxidative phosphorylation of adenosine diphosphate (ADP) to

93 make adenosine triphosphate (ATP) . ATP as well as CO2 and H2O are the final products of the tricarboxylic acid cycle (Krebs cycle) plus oxidative phosphorylation in the mitochondria. Uncoupling agents, such as 2,4-dinitrophenol (DNP), decrease the formation of high-energy phosphate bonds and stimulate systemic oxygen consumption in mitochondria94. The majority of energy-rich phosphate bonds are produced during the step of oxidative phosphorylation, however, in the process of glycolysis, there is a net production of two ATP molecules. During the final phase of cellular energy production,

ATP synthase converts ADP to ATP with the addition of inorganic phosphate.

Mitochondrial uncouplers allow protons to flow across the inner mitochondrial membrane without generating ATP. In their uncharged states, uncouplers are able to dissociate a proton, and in their charged states are able to take up a proton in their pH range of uncoupling. Effective uncouplers generally have a pKa above 6, and less

95 effective uncouplers have pKas below 6 . Proton ionophores, such as DNP or TCS, are neutral or lipophilic when protonated. The export of protons (H+) across the inner mitochondrial membrane is needed for ATP production. Protonated uncoupling chemicals are lipophilic and slip through the inner mitochondrial membrane, bringing protons into the mitochondrial matrix, thus dissipating the electrochemical proton

gradient in which energy from electron transport is stored. The collapse of the essential proton gradient is characteristic of mitochondrial uncouplers.

Uncoupling the electron transport chain system from the process of oxidative phosphorylation can be potentially lethal. Oxidative phosphorylation slows or halts in an uncoupled system, stressing the electron transport chain to work furiously in an attempt to regenerate ATP. While no ATP is generated, oxygen consumption continues to increase. Thus, two key tests for mitochondrial uncoupling include testing for cellular

ATP depletion and an increase in oxygen consumption 96,97.

1.5.1 Effects of Known Mitochondrial Uncouplers

At the turn of the twentieth century, phenol based 2,4-dinitrophenol (DNP), was popularized as an effective weight-loss agent, and is now recognized as a mitochondrial uncoupler98. Due to a high rate of adverse effects and 62 documented deaths attributed to

DNP use, the weight loss drug was recognized as systemically toxic and was banned in the U.S. as a weight loss supplement. Classic symptoms of toxicity included hyperthermia, tachycardia, tachypnoea, diaphoresis and eventually, death98. The shift in the electrochemical gradient of protons due to mitochondrial uncouplers, as previously mentioned (“Uncoupling of Oxidative Phosphorylation”), results in potential energy dissipation as heat instead of conversion to ATP. This process rapidly consumes calories, allowing for weight loss. In the case of DNP used as a weight loss aid, this heat production resulted in a failure of thermoregulatory homeostasis and eventual, uncontrollable hyperthermia.

In contrast to the negative effects of DNP in humans, the 33 kDa inner membrane mitochondrial uncoupling protein, thermogenin, exclusive to brown adipose tissue

(“brown fat”) in mammals is also a proton transporter which uncouples oxidative phosphorylation, dissipating heat of the proton gradient in the respiratory chain99. In response to cold or chronic overeating, heat production, or thermogenesis, in brown fat is activated. Thermogenesis of brown fat in rodents has been shown to play an integral role in thermoregulation and energy balance, and manipulation of this process may prove to be effective against obesity100.

Two proton ionophores, CCCP and carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP), are known mitochondrial uncouplers shown to modulate RBL-

2H3 function53,81,101 and to suppress degranulation. In one study, CCCP was shown to depolarize the plasma membrane in glucose-containing media and to decrease intracellular ATP in glucose-free media93. An evident correlation is shown between the effect of a decrease in ATP production and dampened degranulation in cells cultured in the absence of glucose. CCCP has multiple effects on RBL mast cell function. Under conditions which do not deplete cellular ATP, CCCP was found to depolarize cells and consequentially inhibit antigen-stimulated calcium uptake81.

As shown in previous published work from our lab, non-cytotoxic doses of TCS disrupt mast cell signaling. TCS affects degranulation in response to stimulation by Ag and calcium ionophore. The studies suggest that a potential TCS target lies downstream of calcium influx across the plasma membrane. In this study, we will explore how a separate mechanism involved in cellular energy production may contribute to TCS’s disruption of mast cell function.

1.6 Previous Studies

As mentioned earlier, the Gosse lab has found that TCS dampens mast cell degranulation, the release of pre-formed mediators, which is considered an early phase allergic response57. A cell-based mitochondrial ToxGlo assay from Promega was used to test potential mast cell mitochondrial dysfunction as a result of TCS exposure.

Preliminary data (Figure 3; Weatherly, Shim, Hashmi, Kennedy, Hess and Gosse, submitted) show that TCS disrupts ATP production in RBL-2H3 cells cultured in glucose-free, galactose-containing media (Figure 3A), with a half maximal effective concentration ( EC50) of 7.5-9.6 µM (95% confidence interval), without causing cytotoxicity (tested up to 25 µM).The same experiments were performed with human

HMC-1.2 cells (Figure 3C), yielding similar results: disrupted ATP production with an

EC50 of 4.2-13.7 µM (95% confidence interval) and no cytotoxicity. Expected results were seen in parallel experiments conducted using known mitochondrial uncoupler,

CCCP (Figures 3B; 3D). As expected, ATP depletion was not observed in experiments utilizing glucose, wherein cells produce sufficient ATP via glycolysis. The reduction in

ATP with no change in plasma membrane integrity, in two cell types, suggests that TCS is a mitochondrial toxicant.

Figure 3: Cytotoxicity and ATP production of RBL-2H3 and HMC-1.2 mast cells exposed to toxicant in glucose or glucose-free media. (A) TCS for 1 hr in the presence of BSA and (B) CCCP for 1.5 hr with no BSA. ATP and cytotoxicity responses were also measured in HMC-1.2 cells in galactose media due to TCS (C) or CCCP (D). Dose-response curves for TCS or CCCP are shown on a logarithmic scale. Error bars represent standard error of the mean of data from at least 3 repeat experiments. Statistical significance was determined by one-way ANOVA and Tukey’spost-hoc test. **p<0.01; ***p<0.001. (Weatherly, Shim, Hashmi, Kennedy, Hess and Gosse, submitted).

. 18

TCS-methyl, which has no ionizable proton, but instead, a methyl group in its place, affects neither degranulation (in stark contrast to TCS’s strong inhibition of degranulation57) nor ATP production (Weatherly, Shim, Hashmi, Kennedy, Hess and

Gosse, submitted). Thus, triclosan’s effects on mast cell function are due, at least in part, to its ionizable proton. Also, 5 µM TCS inhibits thapsigargin-stimulated degranulation of

RBL-2H3 cells: further evidence that TCS disrupts mast cell signaling and Ca2+ influx.

As a proton ionophore, TCS likely affects numerous cell processes which depend on electrochemical gradients, in diverse cell types. Also, in mast cells, mitochondria play a role in Ca2+ influx101,102 and ROS production, both of which are needed for cytokine production, and have been shown to be disrupted by other proton ionophores103.

Depletion of ATP due to mitochondrial uncoupling leads to increased fuel oxidation and electron transport in an effort to regenerate the proton gradient93. Since

TCS has been shown to inhibit ATP production in a non-cytotoxic manner, we hypothesize that the rate of consumption of O2, the final electron acceptor in the electron transport chain, will rise in the presence of TCS. Oxygen consumption rate (OCR) is a parameter that has been used to study mitochondrial function, since most cellular oxygen consumption is via mitochondria104. In this study, we measured the OCR as well as examined change in membrane potential to test the aforementioned hypotheses. If TCS is found to be a mitochondrial uncoupler, this mechanism could underlie TCS cytotoxicity in diverse mammalian cell types of many species.

2 Materials and Methods

2.1 Cell Culture

Rat Basophilic Leukemia (RBL) cells, clone 2H3, were provided by D. Holowka

(Cornell University, Ithaca, NY, USA). Cell culture methods were those previously established in the literature105. RBL cells were screened for mycoplasma contamination roughly every 6 weeks using a MycoAlert Mycoplasma detection kit (Lonza).

2.2 Reagents and Buffers

2.2.1 TCS media preparation

Triclosan (TCS) was purchased from Sigma Aldrich (“irgasan,” ≥97% by HPLC).

A 70 µM TCS stock solution was prepared on each experimental day by dissolving TCS in 100 mL of cell culture water (Bio Whittaker). Effective dissolution methods which avoid the use of organic solvents were used58. A cell culture water control stock was prepared similarly alongside the TCS. Concentration was determined via UV-Vis spectrophotometry. Absorbance was recorded at 280 nm, and final TCS concentration was determined using a 1 cm path length, a 4200 M-1Lcm-1extinction coefficient of TCS, and the Beer-Lambert equation. The following media components were measured and added at 39°C, while stirring, to both TCS and control media and thoroughly mixed: 8.3 g/L Dulbecco’s Modified Eagle’s Medium powder without glucose, L-glutamine, phenol red, sodium pyruvate, and sodium bicarbonate (Sigma, D5030-10L), 10 mM D-(+)- galactose (Sigma, G5388-100G), 3.7 g/L sodium bicarbonate (J.T. Baker, 3509-01), and

0.584 g/L L-glutamine (Lonza from VWR, 17-605E). The pH was adjusted to 7.4 using concentrated HCl (BDH), and bovine serum albumin (BSA; Calbiochem) was added.

TCS stock solution was prepared as described above for Mitochondrial Membrane

Potential (MMP) assays beginning with a 100 µM TCS stock solution. TCS was dissolved in Tyrodes Buffer (see 2.2.4) in place of cell culture water and no additional media components were added.

2.2.2 CCCP stock preparation

Carbonyl cyanide m-chlorophenylhydrazone (CCCP), purchased from VWR, was dissolved in 100% DMSO to produce a 100 mM stock solution that was stored at -20°C until the day of use. Glucose-free, galactose-containing media composed of 8.3 g/L

Dulbecco’s Modified Eagle’s Medium, 10 mM D-(+)-galactose (Sigma), 0.584 g/L L- glutamine (Lonza), and 3.7 g/L sodium bicarbonate (VWR) was freshly prepared in 50 mL of cell culture water on the experimental day. CCCP stock was diluted in galactose media to generate a 200 µM CCCP working stock (0.2% DMSO) from which dilutions of

0 µM, 1 µM, and 3 µM were made. Previous control experiments have shown that the tested concentrations of DMSO are not cytotoxic (data not shown).

For mitochondrial membrane potential assays, CCCP positive control is provided by the kit supplier as a 2 mM stock solution in DMSO.

2.2.3 Phenol-red free media

Phenol-red free media was used to plate RBL cells 18- 20 hours prior to assaying oxygen consumption. Phenol-red free minimum essential medium (MEM) without L-

glutamine (Quality Biological) was used in order to minimize background fluorescence due to phenol red pH indicator. Fetal Bovine Serum (FBS) from Atlanta Biologicals (Lot

# F13098), stored at -20°C was thawed for four hours in a 37°C water bath prior to making the media. The same lot of FBS was used throughout associated assays for consistency. All media preparation was performed in a SterilGard tissue culture hood sterilized with 70 % ethanol. FBS, pre-warmed to aid in filtering, was measured out using two 50 mL conical tubes for a total volume of 100 mL which was then added to

400 mL of MEM. Room temperature gentamicin sulfate antibiotic (Lonza) was added to

500 mL of media with a sterile pipette. For a final concentration of 2 mM, 5 mL of 200 mM L-Glutamine (Lonza) was then added to the media. Media was filtered into a sterile, glass, tissue culture media bottle that had previously been autoclaved. A new VacuCap

60 filter with 0.2 µM Supor Membrane (Pall Corporation) was used for vacuum filtration.

2.2.4 Tyrodes Buffer

Tyrodes buffer was composed of 135 mM NaCl (EMD Millipore), 5 mM KCl (JT

Baker), 1.8 mM CaCl2 (ACROS Organics), 1 mM MgCl2, 5.6 mM D-(+)-glucose (MP

BIO), and 20 mM HEPES sodium salt (JT Baker). Stock solutions of NaCl, KCl, MgCl2 and CaCl2 were created in advance in 1M or 0.5 M concentrations, sterile filtered using a

0.2 µM VacuCap filter and stored at 4°C. Buffer components were dissolved in 1 L of ultra-pure Milli-Q water, thoroughly mixed, and sterile filtered. The pH was adjusted to

7.4 and the buffer was stored at 4°C until the day of use.

2.3 Mitochondrial Membrane Potential Assays

Mitochondrial membrane potential was assayed using a Mito-ID Membrane potential cytotoxicity kit for microplates (Enzo Life Sciences). This kit uses a cationic dual-emission dye. In normal cells, the inner mitochondrial membrane is highly polarized and the dye undergoes electrophoretic uptake into the negatively charged inner mitochondrial matrix. The dye fluoresces orange under these normal/polarized conditions. In contrast, in cells whose mitochondria have low membrane potential, e.g., due to mitochondrial uncoupling, the dye remains in the cytosol and fluoresces green106.

The kit was used according the manufacturer’s instructions, using the rapid kinetics measurement, with the following modifications. RBL cells were plated in a black-walled, clear-bottom 96-well microplate (Greiner Bio-One) at a density of 50,000 cells per well. Cells were incubated overnight (16-18 h) in plain RBL media in 5% CO2 at 37°C. Cells were plated for duplicate samples of positive and negative controls and four concentrations of toxicant.

Mito-ID Dye loading solution was prepared by adding 100 μL of light sensitive

Mito-ID MP Detection Reagent, 1 mL 10X Assay Buffer 1 and 0.2 mL of 50X Assay

Buffer 2, supplied by the kit, into 8.7 mL deionized water. Reagents were thoroughly mixed and unused aliquots of 1.3mL were wrapped in aluminum foil and stored in microcentrifuge tubes at -20°C until the day of use.

On the experimental day, 1mg/mL BSA in Tyrodes Buffer was added to make

Tyrodes-BSA buffer (BT) and placed in a 37°C water bath of previously autoclaved milli-Q water. CCCP (2 mM stock) was diluted in BT to generate a 44 µM CCCP

working stock (2.2% DMSO), which served as the positive control. Four TCS concentrations diluted in BT (0.003 µM, 15 µM, 45 µM, and 75 µM)were tested. TCS dilutions were made 3X the desired final concentrations. The prepared microplates containing cells were observed under a microscope for 80-100% confluence. Spent media was discarded in a waste receptacle and cells were washed twice with 200 µL control BT. A volume of 100 µL of BT was added into each well, followed by 100 µL of the prepared Mito-ID Dye Loading Solution directly into the BT. The plate was incubated at room temperature for 30 minutes and shielded from light. To the respective duplicate wells, 100 µL of TCS dilutions and 20 µL of 44 µM CCCP (final concentration of 4 µM,

0.2% DMSO) were added. The bottom of the plate was wiped with a KimWipe and fluorescence measurements of the dye were monitored for 60 minutes via a fluorescent microplate reader (BioTek; Synergy). The dye was excited at 490 nm and fluorescence intensity was measured at 590 nm at normal speed and a sensitivity of 40. The plate was read from the bottom with a topical offset of 7 mm. A second read was conducted with the same setting, but at an emission of 528/20 nm in order to calculate a ratio of orange to green fluorescence.

The above procedure was followed without cells in order to perform a no-cell control experiment.

2.4 Oxygen Consumption Rate Assays

Oxygen consumption rate (OCR) was assayed using an OCR Assay Kit

(MitoXpress-Xtra HS Method) from Cayman Chemical (Ann Arbor, Michigan). This kit uses a MitoXpress-Xtra phosphorescent oxygen probe to measure the OCR in living

cells. The phosphorescence of the kit’s MitoXpress probe is quenched by oxygen such that the emission measured is inversely proportional to the amount of oxygen present in the wells (i.e. an increase in emission indicates oxygen depletion)104.

Prior to use, the MitoXpress probe was centrifuged to localize the contents at the bottom of the vial and then reconstituted in 1 mL of sterile, cell culture water. The vial was vortexed for 30 seconds, followed by a 30 minute sonication step to ensure dissolution of the lyophilized reagent. The reconstituted probe was then vortexed for 30 seconds and centrifuged again before aliquots of 50 µL were prepared in microcentrifuge tubes, wrapped in parafilm and aluminum foil, and stored at -20°C until the experimental day.

The OCR assay was performed in standard mode as per the manufacturer’s instructions with the following changes. Cells were plated in phenol-red free media at a density of 50,000 cells per well in a black, clear-bottom 96-well plate. Cells were incubated overnight (16-18 h) in 5% CO2 and 37°C. The following day, all equipment was pre-warmed so that each step in the OCR assay was performed at 37-39°C. The bacterial incubator, heat blocks, water baths, hot plates for TCS dissolution, and the fluorescence microplate reader were pre-warmed and all micropipettes and disposables were warmed in a 39°C bacterial incubator. Master mixes of the test compound (TCS or

CCCP) were prepared with MitoXpress probe in glucose-free, galactose containing media

(without BSA for CCCP). All dilutions were prepared in pre-warmed conical tubes and microcentrifuge tubes placed in corresponding heat blocks. Master mixes were vortexed, inverted, and spun down in a microcentrifuge. Spent media in the 96-well plate was discarded and wells were washed once with galactose media. The plate was placed on a

96-well plate heat block and a volume of 420 µL of each sample was added to corresponding wells. Bubbles, which trap oxygen and may cause error in the experiment, were carefully popped. Just enough sample volume was added to slightly bubble above the wells without resulting in overflow to allow for a flush seal when the plate was sealed with an oxygen impermeable aluminum plate sealer (VWR). The plate sealer works to effectively trap external oxygen from entering the wells. The plate was inserted into the fluorescence microplate reader set at 37°C and left to incubate for 4 minutes before the plate was read. The phosphorescent probe was excited with a 360/40 nm excitation filter set and emission intensity was measured via a 645/15 nm emission filter. Light emission was read from the bottom of the plate at normal speed with a sensitivity setting of

90.Emission was monitored for 3 hours at 3 minute intervals. Due to the sensitivity of this assay, stringent plate reader parameters were set specifically for the Greiner Bio-One black, clear bottom 96 well plate (Cat no. 655090; Figure #4).

Figure 4: Instrument Signal Optimization Plate reader settings determined for Greiner Bio-One black, clear bottom 96 well plate. Bottom Right X is the distance from the left side of the plate to the center of the last well on the plate (A-G from diagram). Bottom Right Y is the distance from the top of the plate to the center of the last well on the plate (B-H from diagram). All dimensions are in millimeters. Schematic adapted from Greiner Bio-One, 2011.

2.5 Glucose Oxidase and Antimycin A Controls

Glucose oxidase (GO) and Antimycin A (AA; in DMSO) (obtained from the

MitoXpress-Xtra HS Method kit from Cayman Chemical, Ann Arbor, Michigan) control experiments were performed according to the procedure described in 2.4 (Oxygen consumption rate assays). Prior to use, GO was reconstituted in 0.2 mL of sterile, distilled water and stored at -20°C where it is stable for up to two months. Antimycin A, an inhibitor of the mitochondrial electron transport chain, was used as a positive control.

Glucose oxidase was used as a reference for oxygen depletion. GO was used in place of cells in the GO control assay.

2.6 Data analyses

Data from OCR assays were analyzed by first averaging the sample replicates.

Corresponding emission of no probe-containing samples(background)was subtracted from probe-containing samples in order to obtain emission due to the probe itself. For example, [0 µM TCS+Probe+Cells] – [0 µM TCS+No Probe+Cells] = 0 µM TCS +Cells.

[0 µM TCS+Probe+No Cells] – [0 µM TCS+No Probe+No Cells] = 0 µM TCS+No

Cells. Next, the no-cell containing controls were subtracted in order to determine the emission due to the depletion of oxygen by the cells. Continuing with the previous example: [0 µM TCS+Cells] – [0 µM TCS+No Cells] = Final 0 µM TCS value. This process was performed at each time point for every TCS concentration. Data were entered into GraphPad Prism software, and slope by linear regression was determined.

Slopes were normalized to 0 µM and graphed in a bar graph format. Significance was obtained via a one-way ANOVA with Tukey’s post-hoc test.

MMP assays were analyzed by plotting raw fluorescence values against TCS concentration in GraphPad. Significance was obtained as described for OCR assays.

3 Results

3.1 TCS Interference in Measuring Mitochondrial Membrane Potential

In order to test effects of TCS on mitochondrial membrane potential in RBL cells, appropriate no-cell control experiments were conducted (see 2.3. “Mitochondrial

Membrane Potential Assays”) to first investigate whether TCS interacts with the cationic

Mito-ID membrane potential dye, provided by Enzo Life Sciences. In the presence of energetic cells (normal/polarized conditions), highly conjugated moieties of the dual- emission Mito-ID dye delocalize a positive charge across the mitochondrial membrane allowing for its electrophoretic uptake into the negatively charged matrix. The formation of J-aggregates upon membrane polarization causes a shift in emission from ~530-590 nm (green) range to ~490 nm (orange)92. It was found that, in the absence of cells, increasing micromolar concentrations of TCS (0, 0.001, 5, 15, and 25 µM TCS) show significant increases in raw fluorescence of the Mito-ID dye. (In contrast, 4 µM CCCP, a control membrane potential perturbation agent107, did not affect Mito-ID dye fluorescence when compared to 0 µM TCS (Figure 5)). However, TCS + BT solution, when measured via UV-Vis spectrophotometry, exhibited no absorbance at 485 nm or

590 nm, where Mito-ID dye is measured. Minimal fluorescence was seen when TCS,

TCS + BT, and BT-only solutions (without Mito-ID dye) were measured at 485 nm excitation and 590 nm emission. No change was seen between fluorescence control measurements of TCS and TCS + BT samples (data not shown), suggesting TCS and/or

BT are not fluorescing at the specified excitation and emission settings for the Mito-ID dye. Control experiments suggest that TCS is most likely interacting with the fluorescent dye, causing it to aggregate in the cytosol. Based on these results, we determined that the

Mito-ID dye cannot be used with TCS to measure mitochondrial membrane potential.

Additional assays which measure MMP commonly utilize similar cationic, fluorescent probes, dependent on J-aggregate formation108, which we conclude are, likely, incompatible with TCS.

Mito-ID No Cells Test

1750 *** 1500

1250

1000

750 Values *** 500 ***

Raw Raw Fluorescence (4 µM) 250

0 0 0.001 5 15 25 CCCP Concentration(µM)

Figure 5: TCS Interference with Mito-ID Membrane Potential Dye Parameter Value Data Set-B Data Set-C Data Set-D Raw fluorescence 0 vs 0.001 values of-2.875 varying micromolar0.4089 concentrationsP > 0.05 of TCS-31.91 treated to 26.16 with Mito 0- IDvs dye5 in a no-cell-162.9 control23.17 assay (590 nm Pemission) < 0.001. TCS-192.0 is most to likely -133.9 causing the dye to aggregate. Significance was determined via one-way ANOVA 0 vs 15 -455.4 64.77 P < 0.001 -484.5 to -426.4 and Tukey’s 0 vspost 25 hoc test in-1381 GraphPad196.4 Prism software.P <***p<0.001. 0.001 -1410 to -1352 0 vs CCCP -9.938 1.413 P > 0.05 -38.97 to 19.10

3.2 Optimization and Establishment of Oxygen Consumption Rate Assays

Previous work done in the Gosse laboratory has determined that non-cytotoxic doses of TCS inhibit ATP production in multiple cell types (Weatherly, Shim, Hashmi,

Kennedy, Hess and Gosse, submitted). Oxygen consumption rate (OCR) assays can be performed to further investigate triclosan’s effects on mitochondrial function: in particular, to assess its ability to uncouple oxidative phosphorylation from ATP

synthesis109. Our protocol for OCR assays performed in RBL cells was based upon the general assay protocol provided by the manufacturer (Cayman Chemical), with various modifications. Measurements were taken in standard fluorescence intensity mode, rather than in time-resolved mode, using a BioTek Synergy 2 Microplate reader. Due to the extreme technical sensitivity of this assay, a series of parameters were tested before the protocol could be optimized (see Figure 6 for a summary of optimization tests). For suitable measurement of MitoXpress probe, plate reader parameters first needed to be set specifically for the assay. A gain setting of 90 was determined to be the most optimal. A

Greiner Bio-One 96 well plate (Cat # 655090) with minimal autofluorescence was chosen for OCR assays and specific plate dimensions (Figure 4) were programmed, along with the optimized plate reader settings, into Gen5 (version 2.04) plate reader software. To ensure full potency of lyophilized probe provided by the manufacturer, MitoXpress probe was reconstituted and sonicated as described in Materials and Methods (section 2.4). For maximal temperature equilibration, to avoid data artifacts, it was determined that all instruments, reagents, buffers and materials must be pre-warmed to ~ 37°C and kept at this temperature throughout the completion of the assay. This helped to minimize a “dip” in initial fluorescence readings due to temperature adjustment of media components within individual wells of the 96 well plate. Another step evaluated in the development of this protocol was the determination of optimal cell density which would provide an even monolayer of confluent cells and the greatest rate (slope) of oxygen consumption.

A range of cell densities were tested and 50,000 cells per well was determined to yield the most optimal oxygen consumption rate based on the aerobic capacity of RBL cells

(Figure 7). In previous studies of TCS effects on ATP production in RBL cells, glucose-

free, galactose-containing media was utilized to avoid ATP production via glycolysis

(Weatherly, Shim, Hashmi, Kennedy, Hess and Gosse, submitted). For consistency and data comparability, the same media conditions were established for OCR assays. In control experiments, OCR measured in cells supplemented with galactose media was higher than in parallel assays using glucose media (data not shown). All serum, media and buffer ingredients were screened for components which yielded high background fluorescence which may dampen fluorescence seen due to the MitoXpress probe. It was determined that phenol-red pH indicator in cell media, and more significantly, the HS

Mineral Oil overlay (provided by the manufacturer) yielded high background fluorescence, which obscured signal due to cellular oxygen consumption (Figure 8).

Lonza MEM without phenol-red, without L-Glutamine and with Earle’s Balanced Salts

(Cat # 10128-658) was incorporated in a phenol red-free media (see Materials and

Methods, section 2.2.3) in which cells were seeded. Various alternative oil overlays were tested for fluorescence and were found to be high in fluorescence at the measured wavelengths, so an oxygen-impermeable aluminum plate sealer (VWR) with minimal fluorescence was utilized as an alternative (Figure 9). Aluminum leaching into samples was not of concern as the extent of leaching is significant only at high temperatures and low pH ranges which lie outside of our experimental parameters110. Master mixes of the test compounds were prepared to eliminate systematic error due to pipetting inconsistencies. Wells were filled with 420 µL total volumes, slightly more than suggested maximum, in order to eliminate the formation of bubbles which may trap external oxygen beneath the adherent plate sealer if wells were not otherwise sealed flush to the top. Furthermore, to most efficiently assess OCR effects, cells were treated

immediately prior to measurement and equilibrated at 37°C for 4 minutes within the microplate reader prior to read. Collectively, all optimization parameters tested were tailored to our system in order to effectively and accurately measure OCR effects in RBL cells.

Parameter Examined Experimental Test Conclusion

Cell density to be Cell densities tested: Cells will be plated at 0.5 x 106 plated in a black, clear 0.5 x 106 cells/well cells/well at a volume of 100 µL bottom 96-well plate 0.4 x 106 cells/well per well for overnight incubation which will produce 80- 0.3 x 106 cells/well (16-20 h) for optimal OCR. 90% confluence on 0.2 x 106 cells/well experimental day and 0.1 x 106 cells/well greatest rate of oxygen Overnight incubation at consumption 37°C, 5% CO2 Temperature Tested: pre-warm Dip in fluorescence readings is equilibration to buffers, assay reagents, decreased by pre-warming all decrease initial dip in pipettes, and pipette tips assay components and strictly OCR fluorescence using the following maintaining a consistent readings equipment: water baths, environment of 37-39°C titer heat blocks, throughout the duration of the incubators, and experiment. microplate reader.

Fluorescence reading Tested: Effectiveness of Phosphorescent probe works variations between probe after adjusting most effectively when replicate wells reagent reconstitution lyophilized probe is centrifuged, methods reconstituted in sterile cell culture water, vortexed for 30 s, sonicated for 30 min, vortexed again for 30 s, centrifuged once again, and stored at -20°C in 50 µL volumes to minimize freeze- thaw cycles and maintain potency of the probe. Master mixes of test samples were created on experimental day to eliminate variation between wells due to pipetting error. High background Tested: Fluorescence Fluorescence of HS Mineral Oil fluorescence resulting controls of all buffer overlay provided by the in failure to detect components and contents manufacturer is too high (4500- increase in emission of wells were performed. 5000 RFU) to detect emission of values upon treatment Varying volumes of phosphorescent probe at the with probe media were tested prescribed settings. Particles of beneath a fixed volume the highly fluorescent oil may of HS Mineral Oil still be settling into media when overlay (e.g. 100 µL oil volumes of media are increased to 275 µL BT; 100 µL oil beneath the overlay. An to 170 µL BT). alternative must be used.

Alternatives to HS Tested: Raw fluorescence All oil overlays tested have high Mineral Oil overlay of various oil overlays RFU and plastic plate sealers which effectively trap and effectiveness of were not effective at blocking oxygen within plastic and aluminum oxygen. An oxygen individual wells, adherent plate sealers. impermeable, aluminum plate allowing for accurate sealer with minimal signal determination autofluorescence or leaching of due to probe. aluminum was utilized. Plate reader parameters Tested: Gain settings of A gain setting of 90 indicated 80, 90, and 100 for highest signal/background ratio. highest Instrument parameters were signal/background ratio adjusted to recognize dimensions in no-cell controls. specific to the Greiner Bio-One Clear Bottom 96-well plate. Use of galactose or Tested: OCR in RBL Linear regression data indicate glucose containing cells in galactose media, higher OCR in galactose media galactose-BT, glucose conditions. These media media, and glucose-BT conditions are consistent with previous ATP assays associated with this study. Probe to media ratio Manufacturer Addition of probe to increase instructions suggest 10 probe/media ratios does not µL probe to 170 µL enhance the signal seen due to media. Ratios were MX probe. Probe volume adjusted to determine if a should, however, remain higher proportion of proportional to cell density. probe would enhance signal. Data analysis 0, 5, and 10 µM TCS Appropriate no-cell and no-probe each require their own controls must be conducted per no-cell and no-probe experimental condition to be controls. subtracted during data analysis so that OCR seen is due to probe.

Figure 6: Optimization of Oxygen Consumption Rate Assay Protocol

Figure 7: RBL Cell Density Optimization

OCR of varying RBL cell densities (50,000, 40,000, 30,000, 20,000, and 10,000 cells/well) was measured (RFUs) to determine a cell density which would yield the greatest OCR. No-cell ± probe controls were also performed.

Figure 8: Fluorescence controls to identify source of high background

Raw fluorescence measurements of buffers, milli-Q water, and cell culture water, and HS Mineral Oil overlay.

Figure 9: Raw fluorescence of various oil overlays

Raw fluorescence values of various oils were compared against BT in order to seek an alternative to the highly fluorescent HS Mineral Oil overlay.

3.3 TCS Effect on Cellular Oxygen Consumption

After optimization to our system, OCR was measured in RBL cells using a

MitoXpress, cell-impermeable, phosphorescent probe according to the Methods described earlier. Representative linear data indicate that RBL cells exposed to 10 µM TCS exhibit an increase in OCR when compared to 0 µM untreated control in glucose-free, galactose-containing media, as shown in Figure 10A. Combined data of three individual experiments with two replicates each indicate a 1.17-fold ± 0.06 (SEM) increase in cellular OCR in RBL cells at 5 µM when compared to the untreated control and a 1.6- fold ± 0.1 (SEM) increase at 10 µM (also compared to 0 µM TCS untreated control;

Figure 11A). Parallel experiments were conducted using 1 µM CCCP and a 0 µM untreated control, also showing an increase in OCR (Figure 10B). A 1.5-fold ± 0.1

(SEM) increase is seen at 1 µM CCCP when compared to the untreated control (Figure

11B). Our data indicate that 10 µM TCS is nearly as potent as 1 µM CCCP. An increase in cellular oxygen consumption, seen during a three hour reading measurement, indicates that the cells remained viable throughout the assay. Upon observation, cells also appeared healthy following the assay.

No-cell control experiments show a slope of zero, or no increase in emission over time, in the presence of probe. Furthermore, the addition of TCS, in the absence of probe, did not cause an artificial increase in fluorescence over time (data not shown).

These OCR data indicate that TCS increases cellular oxygen consumption, similar to the known mitochondrial uncoupler CCCP. Taken together, TCS’s effects on disrupting

ATP production and increasing cellular OCR further indicate that it is a mitochondrial uncoupler.

Figure 10: Representative linear data of OCR in RBL cells

RBL cellular OCR was measured in the presence of 10 µM TCS (A) and 1 µM CCCP (B) and corresponding 0 µM untreated controls. Data representative of individual experiments were plotted in GraphPad Prism software.

Figure 11: Slope of OCR effects due to toxicant exposure

OCR was measured in glucose-free, galactose-containing media in RBL cells. Effects of 5 µM and 10 µM TCS were compared to a 0 µM untreated control. Effects of 1 µM CCCP, a positive control for this assay, were compared to 0 µM untreated control. Values depicted represent the mean ± SEM of (A) three independent experiments (B) five independent experiments; two replicates per experiment. Significance was determined via one-way ANOVA and Tukey’spost hoc test in GraphPad Prism software. **p<0.01

3.4 Validation that the Increase in OCR is not due to Toxicant Interaction

with Phosphorescent Oxygen Probe

In order to validate the results of our OCR data, respiration of RBL cells was measured in the presence and absence of an electron transport chain inhibitor, Antimycin

A (AA). AA inhibits electron transport beyond complex III in the electron transport chain111. In the presence of AA, RBL cells exhibited no increase in respiration. AA blocks any increase of OCR by TCS in RBL cells (Figure 12).

To further confirm that the increase in oxygen consumption is due to cellular depletion of oxygen and not due to interaction of toxicant with the phosphorescent probe, glucose oxidase was used as a reference for oxygen depletion. In a no-cell experiment, it was determined that 5 and 10 µM TCS do not affect emission due to the phosphorescent probe in a deoxygenated environment (Figure 13A). Parallel experiments were conducted with 1 µM CCCP control and a slight dampening in emission was seen (Figure

13B). This dampening, possibly due to toxicant interaction with the probe or with glucose oxidase, would, however, only lead to a slight underestimation of CCCP effects on OCR.

Figure 12: OCR effects in RBL cells in the presence of ± Antimycin A (AA) and ± TCS OCR was measured in RBL cells in the presence of electron transport chain inhibitor, Antimycin A under the following experimental conditions: 0 µM TCS

+ AA and 10 µM TCS + AA with corresponding no AA-containing controls. Values represent the mean ± SEM of three independent experiments; two replicates per experiment. Significance was determined via one-way ANOVA and Tukey’spost hoc test in GraphPad Prism software. ***p<0.001; *p<0.05.

Figure 13: OCR Measured in Oxygen Depleted, No-Cell Environment

Glucose oxidase was added to master mixes containing TCS (A) or CCCP (B) and OCR was measured (as described in Materials and Methods) in the absence of cells; two replicates per experiment. Raw Fluorescence Units (RFU) displayed are means ± SD of two independent experiments per condition (TCS or CCCP).

4 Discussion

4.1 Discussion of Mitochondrial Membrane Potential Assay

The energy inherent in the electrochemical proton gradient built by the electron transport chain within the inner mitochondrial membrane drives the synthesis of ATP.

By dissipating the proton gradient, mitochondrial uncouplers disrupt the tight coupling between electron transport and oxidative phosphorylation. Pharmacologic uncoupling by these hydrophobic molecules, which possess dissociable protons, causes depolarization across the mitochondrial membrane112. In normal cells, the inner mitochondrial membrane is highly polarized93. Proton ionophores have been shown to decrease mitochondrial membrane potential 113. Due to potential TCS interference with the cationic, dual-emission membrane potential dye provided by Enzo Life Sciences, we were unable to measure fluctuations in MMP due to TCS. Characteristic of most mitochondrial probes is the formation of fluorescent J-aggregates within the cytosol or within mitochondria (described in “Results”). Figure 1 depicts a no-cell control experiment where TCS treated with Mito-ID MMP dye shows a significant increase in fluorescence due to increasing concentrations of TCS, which, in the presence of cells, would have suggested false hyperpolarization. If TCS is indeed interacting with the

Mito-ID dye, such methods are ineffective for measuring TCS effects on MMP.

Pendergrass et al, has examined the efficacy of such lipophilic, cationic fluorescent dyes which measure changes in MMP in living cells114. Although the study reports confidence in the use of MMP dyes such as Chloromethyl-X-rosamine (CMXRos) and MitoTracker

Green (MTG), they caution investigators to determine that observations gathered from experiments relate well to the experimental system and not to erroneous dye effects114.

Studying the impact of environmental chemicals on mitochondrial function is a means of predicting exposure related toxicity115. Using a high throughput screening

(qHTS) approach, Attene-Ramos et al found that in HepG2 cells, TCS decreased mitochondrial membrane potential115: further evidence that uncoupling effects of TCS are due to its ionizable proton. In cells utilizing glycolysis for their energy needs, other proton ionophores have been shown to depolarize the mitochondrial and plasma membranes81,113, reduce calcium influx across the plasma membrane81,113, and also increase calcium efflux from the mitochondria113.

In mast cells, mitochondrial membrane dysfunction, namely, collapse of transmembrane potential, has been shown to suppress calcium-dependent pro- inflammatory mediator release101.The dissipation of this proton gradient and resultant collapse of the proton motive force prevents calcium influx by inactivating the Ca2+ release-activated Ca2+ (CRAC) current. Because calcium intake and resultant mediator release is dependent upon membrane potential, the presence of an uncoupler can severely impair numerous cell processes which depend on electrochemical gradients113,116.

Furthermore, basic Henderson-Hasselbalch calculations (along with pH values for the mitochondrial intermembrane space and matrix117) indicate that approximately 91% of TCS is protonated in the mitochondrial intermembrane space and, thus, is lipophilic and can enter the mitochondrial matrix. In the higher-pH matrix, ~45% of TCS molecules are deprotonated, charged, hydrophilic, and trapped in the matrix, thus shedding protons on the matrix side, decreasing the transmembrane proton motive force for making ATP. We suggest that the resultant amount of TCS getting across the inner

mitochondrial membrane of cells exposed to micromolar levels of TCS can have substantial effects on uncoupling and mitochondrial dysfunction due to TCS.

In rat thymocytes, 3 μM TCS was found to cause effects on polarization of the plasma membrane of intact membranes of living cells 118. Bis-(1,3-dibutylbarbituric acid) trimethineoxonol (di-BA-C4) fluorescent dye was used to examine TCS effects. In order to further examine the basis of TCS immunotoxicity, future experiments in the Gosse lab may examine TCS effects on plasma membrane potential in mast cells. TCS may be affecting other cellular functions which are dependent on electrochemical gradients in numerous cell types. Taken together, these mechanisms of action could help explain

TCS effects on inhibition of degranulation in mast cells.

4.2 Discussion of Oxygen Consumption Rate Assays

Under glucose-free, galactose-fed conditions which restrict mast cells to oxidative phosphorylation93,119, it was previously found that TCS inhibits ATP production in RBL-

2H3 and HMC-1.2 mast cell lines (Figure 3; Weatherly, Shim, Hashmi, Kennedy, Hess and Gosse, submitted). In this study, our findings show that TCS increases oxygen consumption rate (OCR) in RBL mast cells (Figure 10A). Our optimized protocol for measuring OCR is fine-tuned for use in a standard fluorescence microplate reader, as opposed to measurement by Extracellular Flux (XF) technology (Seahorse Bioscience) specific for OCR or extracellular acidification rate measurements120. In our system, OCR was shown to significantly increase with 10 µM TCS, corresponding well within the EC50 range determined for ATP depletion by TCS (Weatherly, Shim, Hashmi, Kennedy, Hess and Gosse, submitted). The micromolar concentrations used in these experiments are

one-thousand times lower than the concentrations found commonly in TCS-containing personal care products 3,4. It is interesting to note that 10 µM TCS exhibited similar effects on RBL mast cell OCR as 1 µM CCCP, noted as a canonical and potent uncoupler of oxidative phosphorylation. We confirmed that these effects are due to cellular depletion of oxygen and not due to toxicant interaction with the phosphorescent probe

(Figure 12 and 13).

Previous studies in the Gosse lab have found TCS to dampen degranulation in

RBL cells in both glucose- and galactose-containing media. Additionally, it has been shown that TCS dampens degranulation in human mast cells and by stimulation methods that bypass FcRI in RBL cells. In addition to previously shown TCS depletion of ATP production at non-cytotoxic doses, a concomitant increase in cellular oxygen consumption supports the hypothesis that TCS is a mitochondrial uncoupler.

Uncouplers have been shown to have antioxidant effects as a result of their reduced proton motive force (PMF) and due to the shortened occupancy of time of single electrons within the electron carriers of the electron transport chain121. Pharmacologic and genetic uncouplers have also been shown to have favorable effects on various disorders linked to oxidative stress122,123, including heart failure124, insulin resistance125,126, ischemic-reperfusion injury112, Parkinson’s disease127, and also diseases such as obesity which may benefit from an increase in energy expenditure128. The varying usages of mitochondrial uncouplers implicate that TCS has potential aside from being an antimicrobial agent. However, due to widespread exposure to TCS via common household and personal care products, it is important to consider the adverse effects of its uncoupling property98.

5 References

1 Levy, C. W. et al. Molecular basis of triclosan activity. Nature 398, 383-384, doi:10.1038/18803 (1999). 2 Adolfsson-Erici, M., Pettersson, M., Parkkonen, J. & Sturve, J. Triclosan, a commonly used bactericide found in human milk and in the aquatic environment in Sweden. Chemosphere 46, 1485-1489 (2002). 3 Jones, R. D., Jampani, H. B., Newman, J. L. & Lee, A. S. Triclosan: A review of effectiveness and safety in health care settings. American Journal of Infection Control 28, 184-196 (2000). 4 Rodricks, J. V., Swenberg, J. A., Borzelleca, J. F., Maronpot, R. R. & Shipp, A. M. Triclosan: a critical review of the experimental data and development of margins of safety for consumer products. Crit Rev Toxicol 40, 422-484, doi:10.3109/10408441003667514 (2010). 5 Perencevich, E. N., Wong, M. T. & Harris, A. D. National and regional assessment of the antibacterial soap market: a step toward determining the impact of prevalent antibacterial soaps. American journal of infection control 29, 281- 283 (2001). 6 Black, J. G., Howes, D. & Rutherford, T. Percutaneous absorption and metabolism of Irgasan DP300. Toxicology 3, 33-47 (1975). 7 Kanetoshi, A. Acute toxicity, percutaneous absorption and effects on hepatic mixed function oxidase activities of 2,4,4'-trichloro-2'-hydroxydiphenyl ether (Irgasan DP300) and its chlorinated derivatives. Environmental Contamination and Toxicology 23, 91-98 (1992). 8 Sandborgh-Englund, G., Adolfsson-Erici, M., Odham, G. & Ekstrand, J. Pharmacokinetics of triclosan following oral ingestion in humans. Journal of toxicology and environmental health. Part A 69, 1861-1873, doi:10.1080/15287390600631706 (2006). 9 Aiello, A. E., Larson, E. L. & Levy, S. B. Consumer antibacterial soaps: effective or just risky? Clinical infectious diseases : an official publication of the Infectious Diseases Society of America 45 Suppl 2, S137-147, doi:10.1086/519255 (2007). 10 Heath, R. J. et al. Mechanism of triclosan inhibition of bacterial fatty acid synthesis. J Biol Chem 274, 11110-11114 (1999). 11 McMurry, L. M., Oethinger, M. & Levy, S. B. Triclosan targets lipid synthesis. Nature 394, 531-532 (1998). 12 Jones, R. D., Jampani, H. B., Newman, J. L. & Lee, A. S. Triclosan: a review of effectiveness and safety in health care settings. American journal of infection control 28, 184-196 (2000). 13 Giuliano, C. A. & Rybak, M. J. Efficacy of triclosan as an antimicrobial hand soap and its potential impact on antimicrobial resistance: a focused review. Pharmacotherapy 35, 328-336, doi:10.1002/phar.1553 (2015).

14 Zafar, A. B., Butler, R. C., Reese, D. J., Gaydos, L. A. & Mennonna, P. A. Use of 0.3% triclosan (Bacti-Stat) to eradicate an outbreak of methicillin-resistant Staphylococcus aureus in a neonatal nursery. American journal of infection control 23, 200-208 (1995). 15 Lanini, S. et al. Molecular epidemiology of a Pseudomonas aeruginosa hospital outbreak driven by a contaminated disinfectant-soap dispenser. PloS one 6, e17064, doi:10.1371/journal.pone.0017064 (2011). 16 Zhu, L., Lin, J., Ma, J., Cronan, J. E. & Wang, H. Triclosan resistance of Pseudomonas aeruginosa PAO1 is due to FabV, a triclosan-resistant enoyl-acyl carrier protein reductase. Antimicrobial agents and chemotherapy 54, 689-698, doi:10.1128/aac.01152-09 (2010). 17 Chuanchuen, R. et al. Cross-resistance between triclosan and antibiotics in Pseudomonas aeruginosa is mediated by multidrug efflux pumps: exposure of a susceptible mutant strain to triclosan selects nfxB mutants overexpressing MexCD-OprJ. Antimicrobial agents and chemotherapy 45, 428-432, doi:10.1128/aac.45.2.428-432.2001 (2001). 18 Freundlich, J. S. et al. Triclosan Derivatives: Towards Potent Inhibitors of Drug- Sensitive and Drug-Resistant Mycobacterium tuberculosis. ChemMedChem 4, 241-248, doi:10.1002/cmdc.200800261 (2009). 19 Allmyr, M., Adolfsson-Erici, M., McLachlan, M. S. & Sandborgh-Englund, G. Triclosan in plasma and milk from Swedish nursing mothers and their exposure via personal care products. Science of the Total Environment 372, 87-93, doi:10.1016/j.scitotenv.2006.08.007 (2006). 20 Calafat, A. M., Ye, X., Wong, L. Y., Reidy, J. A. & Needham, L. L. Urinary concentrations of Triclosan in the US population: 2003-2004. Environmental Health Perspectives 116, 303-307, doi:10.1289/ehp.10768 (2008). 21 Okumura, T. & Nishikawa, Y. Gas chromatography mass spectrometry determination of triclosans in water, sediment and fish samples via methylation with diazomethane. Analytica Chimica Acta 325, 175-184, doi:10.1016/0003- 2670(96)00027-x (1996). 22 Coogan, M. A., Edziyie, R. E., La Point, T. W. & Venables, B. J. Algal bioaccumulation of triclocarban, triclosan, and methyl-triclosan in a North Texas wastewater treatment plant receiving stream. Chemosphere 67, 1911-1918, doi:10.1016/j.chemosphere.2006.12.027 (2007). 23 Latch, D. E. et al. Aqueous photochemistry of triclosan: Formation of 2,4- dichlorophenol, 2,8-dichlorodibenzo-p-dioxin, and oligomerization products. Environmental Toxicology and Chemistry 24, 517-525, doi:10.1897/04-243r.1 (2005). 24 Reiss, R., Mackay, N., Habig, C. & Griffin, J. An ecological risk assessment for triclosan in lotic systems following discharge from wastewater treatment plants in

the United States. Environmental Toxicology and Chemistry 21, 2483-2492, doi:10.1897/1551-5028(2002)021<2483:aeraft>2.0.co;2 (2002). 25 Singer, H., Muller, S., Tixier, C. & Pillonel, L. Triclosan: occurrence and fate of a widely used biocide in the aquatic environment: field measurements in wastewater treatment plants, surface waters, and lake sediments. Environmental science & technology 36, 4998-5004 (2002). 26 Lindstrom, A. et al. Occurrence and environmental behavior of the bactericide triclosan and its methyl derivative in surface waters and in wastewater. Environ Sci Technol 36, 2322-2329 (2002). 27 Bester, K. Triclosan in a sewage treatment process--balances and monitoring data. Water research 37, 3891-3896, doi:10.1016/s0043-1354(03)00335-x (2003). 28 Heidler, J. & Halden, R. U. Mass balance assessment of triclosan removal during conventional sewage treatment. Chemosphere 66, 362-369, doi:http://dx.doi.org/10.1016/j.chemosphere.2006.04.066 (2007). 29 Balmer, M. E. et al. Occurrence of methyl triclosan, a transformation product of the bactericide triclosan, in fish from various lakes in Switzerland. Environ Sci Technol 38, 390-395 (2004). 30 Agency, U. S. E. P. Pesticides: Reregistration, (2015). 31 Bhargava, H. N. & Leonard, P. A. Triclosan: Applications and safety. American Journal of Infection Control 24, 209-218 (1996). 32 Dann, A. B. & Hontela, A. Triclosan: environmental exposure, toxicity and mechanisms of action. Journal of applied toxicology : JAT 31, 285-311, doi:10.1002/jat.1660 (2011). 33 Calafat, A. M., Ye, X., Wong, L. Y., Reidy, J. A. & Needham, L. L. Urinary concentrations of triclosan in the U.S. population: 2003-2004. Environ Health Perspect 116, 303-307, doi:10.1289/ehp.10768 (2008). 34 Tan, W. P., Suresh, S., Tey, H. L., Chiam, L. Y. & Goon, A. T. A randomized double-blind controlled trial to compare a triclosan-containing emollient with vehicle for the treatment of atopic dermatitis. Clin Exp Dermatol 35, e109-112 (2010). 35 Breneman, D. L., Hanifin, J. M., Berge, C. A., Keswick, B. H. & Neumann, P. B. The effect of antibacterial soap with 1.5% triclocarban on Staphylococcus aureus in patients with atopic dermatitis. Cutis 66, 296-300 (2000). 36 Schnopp, C., Ring, J. & Mempel, M. The role of antibacterial therapy in atopic eczema. Expert opinion on pharmacotherapy 11, 929-936, doi:10.1517/14656561003659992 (2010). 37 Ahn, K. C. et al. In vitro biologic activities of the antimicrobials triclocarban, its analogs, and triclosan in bioassay screens: receptor-based bioassay screens. Environ Health Perspect 116, 1203-1210 (2008).

38 Liu, B., Wang, Y., Fillgrove, K. L. & Anderson, V. E. Triclosan inhibits enoyl- reductase of type I fatty acid synthase in vitro and is cytotoxic to MCF-7 and SKBr-3 breast cancer cells. Cancer chemotherapy and pharmacology 49, 187- 193, doi:10.1007/s00280-001-0399-x (2002). 39 Koeppe, E. S., Ferguson, K. K., Colacino, J. A. & Meeker, J. D. Relationship between urinary triclosan and paraben concentrations and serum thyroid measures in NHANES 2007-2008. Sci Total Environ 445-446, 299-305, doi:10.1016/j.scitotenv.2012.12.052 (2013). 40 Barkvoll, P. & Rolla, G. Triclosan protects the skin against dermatitis caused by sodium lauryl sulfate exposure. Journal of Dental Research 74, 476-476 (1995a). 41 Waaler, S. M., Rolla, G., Skjorland, K. K. & Ogaard, B. Effects of oral rinsing with triclosan and sodium lauryl sulfate on dental plaque-formation - A pilot study. Scandinavian Journal of Dental Research 101, 192-195 (1993). 42 Udoji, F., Martin, T., Etherton, R. & Whalen, M. M. Immunosuppressive effects of triclosan, nonylphenol, and DDT on human natural killer cells in vitro. Journal of Immunotoxicology 7, 205-212, doi:10.3109/15476911003667470 (2010). 43 Sonneveld, E., Jansen, H. J., Riteco, J. A., Brouwer, A. & van der Burg, B. Development of androgen- and estrogen-responsive bioassays, members of a panel of human cell line-based highly selective steroid-responsive bioassays. Toxicological sciences : an official journal of the Society of Toxicology 83, 136- 148, doi:10.1093/toxsci/kfi005 (2005). 44 Chen, J. et al. Antiandrogenic properties of parabens and other phenolic containing small molecules in personal care products. Toxicol Appl Pharmacol 221, 278-284 (2007). 45 Larsson, A., Eriksson, L. A., Andersson, P. L., Ivarson, P. & Olsson, P. E. Identification of the brominated flame retardant 1,2-dibromo-4-(1,2- dibromoethyl)cyclohexane as an androgen agonist. Journal of medicinal chemistry 49, 7366-7372, doi:10.1021/jm060713d (2006). 46 Foran, C. M., Bennett, E. R. & Benson, W. H. Developmental evaluation of a potential non-steroidal estrogen: triclosan. Mar Environ Res 50, 153-156 (2000). 47 Crofton, K. M., Paul, K. B., Devito, M. J. & Hedge, J. M. Short-term in vivo exposure to the water contaminant triclosan: Evidence for disruption of thyroxine. Environmental toxicology and pharmacology 24, 194-197, doi:10.1016/j.etap.2007.04.008 (2007). 48 Wang, L. Q., Falany, C. N. & James, M. O. Triclosan as a substrate and inhibitor of 3'-phosphoadenosine 5'-phosphosulfate-sulfotransferase and UDP-glucuronosyl transferase in human liver fractions. Drug Metab Dispos 32, 1162-1169 (2004). 49 Barros, S. P. et al. Triclosan inhibition of acute and chronic inflammatory gene pathways. Journal of clinical periodontology 37, 412-418, doi:10.1111/j.1600- 051X.2010.01548.x (2010).

50 Gaffar, A., Scherl, D., Afflitto, J. & Coleman, E. J. The effect of triclosan on mediators of gingival inflammation. Journal of clinical periodontology 22, 480- 484 (1995). 51 Whalen, M. M., Wilson, S., Gleghorn, C. & Loganathan, B. G. Brief exposure to triphenyltin produces irreversible inhibition of the cytotoxic function of human natural killer cells. Environmental research 92, 213-220 (2003). 52 Hinkson, N. C. & Whalen, M. M. Hexabromocyclododecane decreases the lytic function and ATP levels of human natural killer cells. Journal of applied toxicology : JAT 29, 656-661, doi:10.1002/jat.1453 (2009). 53 Kuby, J. Immunology. Sixth edn, (W.H. Freeman, New York, 1997). 54 Kookana, R. S., Ying, G. G. & Waller, N. J. Triclosan: its occurrence, fate and effects in the Australian environment. Water science and technology : a journal of the International Association on Water Pollution Research 63, 598-604, doi:10.2166/wst.2011.205 (2011). 55 McDonnell, G. & Russell, A. D. Antiseptics and disinfectants: Activity, action, and resistance. Clinical Microbiology Reviews 12, 147-+ (1999). 56 Newton, A. P. N., Cadena, S., Rocha, M. E. M., Carnieri, E. G. S. & de Oliveira, M. B. M. Effect of triclosan (TRN) on energy-linked functions of rat liver mitochondria. Toxicology Letters 160, 49-59, doi:10.1016/j.toxlet.2005.06.004 (2005). 57 Palmer, R. K. et al. Antibacterial agent triclosan suppresses RBL-2H3 mast cell function. Toxicol Appl Pharmacol 258, 99-108 (2012). 58 Weatherly, L., Kennedy, R., Shim, J., Gosse, J. A Microplate Assay to Assess Chemical Effects on RBL-2H3 Mast Cell Degranulation: Effects of Triclosan without Use of an Organic Solvent. J. Vis. Exp. (2013). 59 Blank, U. et al. SNAREs and associated regulators in the control of exocytosis in the RBL-2H3 mast cell line. Mol Immunol 38, 1341-1345 (2002). 60 Farrell, D. J. et al. Intrahepatic mast cells in chronic liver diseases. Hepatology 22, 1175-1181 (1995). 61 Silver, R. & Curley, J. P. Mast cells on the mind: new insights and opportunities. Trends Neurosci 36, 513-521 (2013). 62 Sigal, L. H. Basic Science for the Clinician 53 Mast Cells. JCR-J. Clin. Rheumatol. 17, 395-400, doi:10.1097/RHU.0b013e31823150b5 (2011). 63 Okayama, Y. & Kawakami, T. Development, migration, and survival of mast cells. Immunol Res 34, 97-115 (2006). 64 Schwartz, L. B. & Austen, K. F. Enzymes of the mast-cell granule. Journal of Investigative Dermatology 74, 349-353, doi:10.1111/1523-1747.ep12543620 (1980).

65 Holgate, S. T. The role of mast cells and basophils in inflammation. Clinical and experimental allergy : journal of the British Society for Allergy and Clinical Immunology 30 Suppl 1, 28-32 (2000). 66 Plaut, M. et al. Mast-cells lines produce lymphokines in response to cross-likage of Fc-epsilon-R1 or to calcium ionophores. Nature 339, 64-67, doi:10.1038/339064a0 (1989). 67 Lowman, M. A., Rees, P. H., Benyon, R. C. & Church, M. K. Human mast cell heterogeneity: histamine release from mast cells dispersed from skin, lung, adenoids, tonsils, and colon in response to IgE-dependent and nonimmunologic stimuli. The Journal of allergy and clinical immunology 81, 590-597 (1988). 68 Lowman, M. A., Rees, P. H., Benyon, R. C. & Church, M. K. Human mast-cell heterogeneity - histamine release from mast-cells dispersed from skin, lung, adenoids, tonsils, and colon in response to IgE-dependent and nonimmunologic stimuli. Journal of Allergy and Clinical Immunology 81, 574-579 (1988). 69 Metcalfe, D. D. Mast cells and mastocytosis. Blood 112, 946-956, doi:10.1182/blood-2007-11-078097 (2008). 70 Passante, E., Ehrhardt, C., Sheridan, H. & Frankish, N. RBL-2H3 cells are an imprecise model for mast cell mediator release. Inflamm. Res. 58, 611-618, doi:10.1007/s00011-009-0028-4 (2009). 71 Maeyama, K., Hohman, R. J., Metzger, H. & Beaven, M. A. Quantitative relationships between aggregation of IgE receptors, generation of intracellular signals, and histamine secretion in rat basophilic leukemia (2H3) cells. Enhanced responses with heavy water. The Journal of biological chemistry 261, 2583-2592 (1986). 72 Seldin, D. C. et al. Homology of the rat basophilic leukemia cell and the rat mucosal mast cell. Proceedings of the National Academy of Sciences of the United States of America 82, 3871-3875 (1985). 73 Metzger, H. et al. The receptor with high-affinity for immunoglobulin-E. Annual Review of Immunology 4, 419-470, doi:10.1146/annurev.immunol.4.1.419 (1986). 74 Fewtrell, C., Kessler, A. & Metzger, H. Comparative aspects of secretion from tumor and normal mast cells. Adv. Inflam. Res 1, 205-221 (1979). 75 Naal, R. M., Tabb, J., Holowka, D. & Baird, B. In situ measurement of degranulation as a biosensor based on RBL-2H3 mast cells. Biosens Bioelectron 20, 791-796 (2004). 76 Ortega, E., Schweitzer-Stenner, R. & Pecht, I. Kinetics of ligand binding to the type 1 Fc epsilon receptor on mast cells. Biochemistry 30, 3473-3483 (1991). 77 Dearman, R. J., Skinner, R. A., Deakin, N., Shaw, D. & Kimber, I. Evaluation of an in vitro method for the measurement of specific IgE antibody responses: the rat basophilic leukemia (RBL) cell assay. Toxicology 206, 195-205, doi:10.1016/j.tox.2004.08.007 (2005).

78 Nilsson, G. et al. Phenotypic characterization of the human mast-cell line HMC-1. Scandinavian Journal of Immunology 39, 489-498, doi:10.1111/j.1365- 3083.1994.tb03404.x (1994). 79 Butterfield, J. H., Weiler, D., Dewald, G. & Gleich, G. J. Establishment of an immature mast-cell line from a patient with mast-cell leukemia. Leukemia Research 12, 345-355, doi:10.1016/0145-2126(88)90050-1 (1988). 80 Butterfield, J. H., Weiler, D., Dewald, G. & Gleich, G. J. Establishment of an immature mast cell line from a patient with mast cell leukemia. Leuk Res 12, 345- 355 (1988). 81 Mohr, F. C. & Fewtrell, C. The relative contributions of extracellular and intracellular calcium to secretion from tumor mast cells. Multiple effects of the proton ionophore carbonyl cyanide m-chlorophenylhydrazone. The Journal of biological chemistry 262, 10638-10643 (1987). 82 Abraham, S. N. & St John, A. L. Mast cell-orchestrated immunity to pathogens. Nat Rev Immunol 10, 440-452 (2010). 83 Burgoyne, R. D. & Morgan, A. Secretory granule exocytosis. Physiol Rev 83, 581-632, doi:10.1152/physrev.00031.2002 (2003). 84 Perez-Montfort, R., Kinet, J. P. & Metzger, H. A previously unrecognized subunit of the receptor for Immunoglobulin-E. Biochemistry 22, 5722-5728, doi:10.1021/bi00294a007 (1983). 85 Siraganian, R. P., de Castro, R. O., Barbu, E. A. & Zhang, J. A. Mast cell signaling: The role of protein tyrosine kinase Syk, its activation and screening methods for new pathway participants. Febs Letters 584, 4933-4940, doi:10.1016/j.febslet.2010.08.006 (2010). 86 Okkenhaug, K. & Vanhaesebroeck, B. PI3K in lymphocyte development, differentiation and activation. Nature Reviews Immunology 3, 317-330, doi:10.1038/nri1056 (2003). 87 Kalesnikoff, J. & Galli, S. J. New developments in mast cell biology. Nature Immunology 9, 1215-1223, doi:10.1038/ni.f.216 (2008). 88 Kraft, S. & Kinet, J. P. New developments in Fc epsilon RI regulation, function and inhibition. Nature Reviews Immunology 7, 365-378, doi:10.1038/nri2072 (2007). 89 Scharenberg, A. M., Humphries, L. A. & Rawlings, D. J. Calcium signalling and cell-fate choice in B cells. Nature Reviews Immunology 7, 778-789, doi:10.1038/nri2172 (2007). 90 Ma, H. T. & Beaven, M. A. Regulators of Ca(2+) signaling in mast cells: potential targets for treatment of mast cell-related diseases? Adv Exp Med Biol 716, 62-90 (2011). 91 Kalesnikoff, J. & Galli, S. J. New developments in mast cell biology. Nat Immunol 9, 1215-1223 (2008).

92 Sciences, E. L. in Enzo Life Scienecs, Inc. (2014). 93 Nelson, D. L., Cox, Michael M., Lehninger, Albert L. Principles of Biochemistry. 6 edn, 690 (W.H. Freeman, 2008). 94 DInitrophenol and accelerated tissue metabolism. Journal of the American Medical Association 101, 2122-2123, doi:10.1001/jama.1933.02740520032013 (1933). 95 Kessler, R. J. et al. Uncouplers and the molecular mechanism of uncoupling in mitochondria. Proceedings of the National Academy of Sciences of the United States of America 74, 2241-2245 (1977). 96 Brand, M. D. & Nicholls, D. G. Assessing mitochondrial dysfunction in cells. Biochem J 435, 297-312, doi:10.1042/bj20110162 (2011). 97 Hanstein, W. G. Uncoupling of oxidative phosphorylation. Biochim Biophys Acta 456, 129-148 (1976). 98 Grundlingh, J., Dargan, P. I., El-Zanfaly, M. & Wood, D. M. 2,4-dinitrophenol (DNP): a weight loss agent with significant acute toxicity and risk of death. Journal of medical toxicology : official journal of the American College of Medical Toxicology 7, 205-212, doi:10.1007/s13181-011-0162-6 (2011). 99 Palou, A., Pico, C., Bonet, M. L. & Oliver, P. The uncoupling protein, thermogenin. The international journal of biochemistry & cell biology 30, 7-11 (1998). 100 Scarpace, P. J., Matheny, M., Borst, S. & Tumer, N. Thermoregulation with age: role of thermogenesis and uncoupling protein expression in brown adipose tissue. Proceedings of the Society for Experimental Biology and Medicine. Society for Experimental Biology and Medicine (New York, N.Y.) 205, 154-161 (1994). 101 Inoue, T., Suzuki, Y. & Ra, C. Epigallocatechin-3-gallate inhibits mast cell degranulation, leukotriene C4 secretion, and calcium influx via mitochondrial calcium dysfunction. Free radical biology & medicine 49, 632-640, doi:10.1016/j.freeradbiomed.2010.05.015 (2010). 102 Gilabert, J. A. & Parekh, A. B. Respiring mitochondria determine the pattern of activation and inactivation of the store-operated Ca(2+) current I(CRAC). The EMBO journal 19, 6401-6407, doi:10.1093/emboj/19.23.6401 (2000). 103 Inoue, T., Suzuki, Y., Yoshimaru, T. & Ra, C. Reactive oxygen species produced up- or downstream of calcium influx regulate proinflammatory mediator release from mast cells: role of NADPH oxidase and mitochondria. Biochimica et biophysica acta 1783, 789-802, doi:10.1016/j.bbamcr.2007.12.004 (2008). 104 Oxygen Consumption Rate Assay Kit (MitoXpress® - Xtra HS Method) (2014). 105 Hutchinson, L. M. et al. Inorganic arsenite inhibits IgE receptor-mediated degranulation of mast cells. J Appl Toxicol 31, 231-241 (2009).

106 Mitochondrial ToxGlo™ Assay Technical Manual, (2014). 107 Goldsby, R. A. & Heytler, P. G. UNCOUPLING OF OXIDATIVE PHOSPHORYLATION BY CARBONYL CYANIDE PHENYLHYDRAZONES. II. EFFECTS OF CARBONYL CYANIDE M- CHLOROPHENYLHYDRAZONE ON MITOCHONDRIAL RESPIRATION. Biochemistry 2, 1142-1147 (1963). 108 Perry, S. W., Norman, J. P., Barbieri, J., Brown, E. B. & Gelbard, H. A. Mitochondrial membrane potential probes and the proton gradient: a practical usage guide. BioTechniques 50, 98-115, doi:10.2144/000113610 (2011). 109 Hynes, J., Natoli, E., Jr. & Will, Y. Fluorescent pH and oxygen probes of the assessment of mitochondrial toxicity in isolated mitochondria and whole cells. Current protocols in toxicology / editorial board, Mahin D. Maines (editor-in- chief) ... [et al.] Chapter 2, Unit 2.16, doi:10.1002/0471140856.tx0216s40 (2009). 110 Fekete, V., Deconinck, E., Bolle, F. & Van Loco, J. Modelling aluminium leaching into food from different foodware materials with multi-level factorial design of experiments. Food additives & contaminants. Part A, Chemistry, analysis, control, exposure & risk assessment 29, 1322-1333, doi:10.1080/19440049.2012.688068 (2012). 111 Belskie, K. M., Van Buiten, C. B., Ramanathan, R. & Mancini, R. A. Reverse electron transport effects on NADH formation and metmyoglobin reduction. Meat science 105, 89-92, doi:10.1016/j.meatsci.2015.02.012 (2015). 112 Sack, M. N. Mitochondrial depolarization and the role of uncoupling proteins in ischemia tolerance. Cardiovascular research 72, 210-219, doi:10.1016/j.cardiores.2006.07.010 (2006). 113 Suzuki, Y., Yoshimaru, T., Inoue, T. & Ra, C. Mitochondrial Ca2+ flux is a critical determinant of the Ca2+ dependence of mast cell degranulation. J Leukoc Biol 79, 508-518, doi:10.1189/jlb.0705412 (2006). 114 Pendergrass, W., Wolf, N. & Poot, M. Efficacy of MitoTracker Green and CMXrosamine to measure changes in mitochondrial membrane potentials in living cells and tissues. Cytometry. Part A : the journal of the International Society for Analytical Cytology 61, 162-169, doi:10.1002/cyto.a.20033 (2004). 115 Attene-Ramos, M. S. et al. Profiling of the Tox21 Chemical Collection for Mitochondrial Function to Identify Compounds that Acutely Decrease Mitochondrial Membrane Potential. Environ Health Perspect 123, 49-56, doi:10.1289/ehp.1408642 (2015). 116 Togo, K. et al. Aspirin and salicylates modulate IgE-mediated leukotriene secretion in mast cells through a dihydropyridine receptor-mediated Ca(2+)

influx. Clinical immunology (Orlando, Fla.) 131, 145-156, doi:10.1016/j.clim.2008.09.008 (2009). 117 Porcelli, A. M. et al. pH difference across the outer mitochondrial membrane measured with a green fluorescent protein mutant. Biochemical and biophysical research communications 326, 799-804, doi:10.1016/j.bbrc.2004.11.105 (2005). 118 Kawanai, T. Triclosan, an environmental pollutant from health care products, evokes charybdotoxin-sensitive hyperpolarization in rat thymocytes. Environmental toxicology and pharmacology 32, 417-422, doi:10.1016/j.etap.2011.08.009 (2011). 119 Benard, G. et al. Multi-site control and regulation of mitochondrial energy production. Biochimica et biophysica acta 1797, 698-709, doi:10.1016/j.bbabio.2010.02.030 (2010). 120 BioScience, S. How XF Technology Works, (2013). 121 Korshunov, S. S., Skulachev, V. P. & Starkov, A. A. High protonic potential actuates a mechanism of production of reactive oxygen species in mitochondria. FEBS letters 416, 15-18 (1997). 122 Cunha, F. M., Caldeira da Silva, C. C., Cerqueira, F. M. & Kowaltowski, A. J. Mild mitochondrial uncoupling as a therapeutic strategy. Current drug targets 12, 783-789 (2011). 123 Lou, P. H. et al. Mitochondrial uncouplers with an extraordinary dynamic range. Biochem J 407, 129-140, doi:10.1042/bj20070606 (2007). 124 Brennan, J. P. et al. Mitochondrial uncoupling, with low concentration FCCP, induces ROS-dependent cardioprotection independent of KATP channel activation. Cardiovascular research 72, 313-321, doi:10.1016/j.cardiores.2006.07.019 (2006). 125 Anderson, E. J. et al. Mitochondrial H2O2 emission and cellular redox state link excess fat intake to insulin resistance in both rodents and humans. The Journal of clinical investigation 119, 573-581, doi:10.1172/jci37048 (2009). 126 Hoehn, K. L. et al. Insulin resistance is a cellular antioxidant defense mechanism. Proceedings of the National Academy of Sciences of the United States of America 106, 17787-17792, doi:10.1073/pnas.0902380106 (2009). 127 Wu, Y. N., Munhall, A. C. & Johnson, S. W. Mitochondrial uncoupling agents antagonize rotenone actions in rat substantia nigra dopamine neurons. Brain research 1395, 86-93, doi:10.1016/j.brainres.2011.04.032 (2011). 128 Tseng, Y. H., Cypess, A. M. & Kahn, C. R. Cellular bioenergetics as a target for obesity therapy. Nature reviews. Drug discovery 9, 465-482, doi:10.1038/nrd3138 (2010).

Author’s Biography

Hina N. Hashmi was born in Houston, Texas on June 20th, 1993. Shortly after birth, her family moved to Veazie, Maine, where she has resided ever since. Hina graduated from Bangor High School in 2011. While at the University of Maine, she remained active throughout her undergraduate years and served as an officer for the

Health Professions Club, the Muslim Students Association, the Student Heritage Alliance

Council, and the Maine Society for Microbiology. She has served on public health and medical outreach projects in rural Maine and in Tanzania.

As a Microbiology major, Hina has received an INBRE Functional Genomics

Fellowship, a Center for Undergraduate Research Fellowship, a Susan Elliot Judd Roxby

Memorial Scholarship, an E. Reeve Hitchner Memorial Scholarship, and the 2014

Rezendes Global Service Scholarship and was invited to participate in the Mini Medical

School program at Maine Medical Center and Tufts University School of Medicine.

Upon graduation, Hina will be applying to medical schools with high hopes of attending in the Fall of 2016.