The University of Maine
DigitalCommons@UMaine
Honors College
Spring 2015
Is the Ubiquitous Antibacterial Agent Triclosan an
Uncoupler of Mammalian Mitochondria
Hina Hashmi
University of Maine - Main
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Hashmi, Hina, "Is the Ubiquitous Antibacterial Agent Triclosan an Uncoupler of Mammalian Mitochondria" (2015). Honors College.
Paper 211.
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IS THE UBIQUITOUS ANTIBACTERIAL AGENT TRICLOSAN AN UNCOUPLER
OF MAMMALIAN MITOCHONDRIA?
By
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 3chlorophenylhydrazone (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 RBL2H3 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.
iv
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
1.6
2
Effects of Known Mitochondrial Uncouplers ............................................. 15
Previous Studies ................................................................................................. 17
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
v
2.6
3
Data analyses ...................................................................................................... 28
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
5
Discussion .......................................................................................................... 46
4.1
Discussion of Mitochondrial Membrane Potential Assay .................................. 46
4.2
Discussion of Oxygen Consumption Rate Assays ............................................. 48
References .......................................................................................................... 50
Author’s Biography .......................................................................................................... 60
vi
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-
1
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 crossresistance to antibiotics associated with consumer use of TCS-containing products9.
2
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
3
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
4
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
5
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.
6
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.
7
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
8
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, βhexosaminidase, and serotonin. To increasing concentrations of DNP-BSA antigen,
9
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
10
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 Tcells (LAT) and PI3K86. PLCγ1is recruited to the membrane by LAT where it is
phosphorylated by Syk. PLCγ1 catalyzes the hydrolysis of phosphatidylinositol 4,5bisphosphate (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
11
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.
12
PLD1
SERCA
PLD2
Figure 2: Schematic of degranulation pathway in mast cells.
Figure adapted from Rachel Kennedy and Jonathon Pelletier.
13
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
make adenosine triphosphate (ATP)93. 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
effective uncouplers have pKas below 695. 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
14
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.
15
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 RBL2H3 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.
16
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.
17
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.
19
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
2.2.1
Reagents and Buffers
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
20
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 Lglutamine (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 L21
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.
22
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
23
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
24
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
25
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).
26
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.
27
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.
28
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 dualemission 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
29
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.
30
Mito-ID No Cells Test
Raw Fluorescence
Values
1750
***
1500
1250
1000
750
***
500
***
250
(4 µM)
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
values
of
varying
micromolar
concentrations
of
TCS
treated
0 vs 0.001
-2.875 0.4089
P > 0.05
-31.91
to 26.16
with Mito-ID
dye
in
a
no-cell
control
assay
(590
nm
emission).
TCS
is
most
0 vs 5
-162.9 23.17
P < 0.001
-192.0 tolikely
-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’spost
hoc
test
in
GraphPad
Prism
software.
***p<0.001.
0 vs 25
-1381
196.4
P < 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
31
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-
32
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
33
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.
34
Parameter Examined
Experimental Test
Conclusion
Cell density to be
plated in a black, clear
bottom 96-well plate
which will produce 8090% confluence on
experimental day and
greatest rate of oxygen
consumption
Temperature
equilibration to
decrease initial dip in
OCR fluorescence
readings
Cell densities tested:
0.5 x 106 cells/well
0.4 x 106 cells/well
0.3 x 106 cells/well
0.2 x 106 cells/well
0.1 x 106 cells/well
Overnight incubation at
37°C, 5% CO2
Tested: pre-warm
buffers, assay reagents,
pipettes, and pipette tips
using the following
equipment: water baths,
titer heat blocks,
incubators, and
microplate reader.
Cells will be plated at 0.5 x 106
cells/well at a volume of 100 µL
per well for overnight incubation
(16-20 h) for optimal OCR.
Fluorescence reading
variations between
replicate wells
Tested: Effectiveness of
probe after adjusting
reagent reconstitution
methods
High background
fluorescence resulting
in failure to detect
increase in emission
values upon treatment
with probe
Dip in fluorescence readings is
decreased by pre-warming all
assay components and strictly
maintaining a consistent
environment of 37-39°C
throughout the duration of the
experiment.
Phosphorescent probe works
most effectively when
lyophilized probe is centrifuged,
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 freezethaw 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.
Tested: Fluorescence
Fluorescence of HS Mineral Oil
controls of all buffer
overlay provided by the
components and contents manufacturer is too high (4500of wells were performed. 5000 RFU) to detect emission of
Varying volumes of
phosphorescent probe at the
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.
35
Alternatives to HS
Mineral Oil overlay
which effectively trap
oxygen within
individual wells,
allowing for accurate
signal determination
due to probe.
Plate reader parameters
Tested: Raw fluorescence
of various oil overlays
and effectiveness of
plastic and aluminum
adherent plate sealers.
Use of galactose or
glucose containing
media
Tested: OCR in RBL
cells in galactose media,
galactose-BT, glucose
media, and glucose-BT
Probe to media ratio
Manufacturer
instructions suggest 10
µL probe to 170 µL
media. Ratios were
adjusted to determine if a
higher proportion of
probe would enhance
signal.
0, 5, and 10 µM TCS
each require their own
no-cell and no-probe
controls.
Data analysis
Tested: Gain settings of
80, 90, and 100 for
highest
signal/background ratio
in no-cell controls.
All oil overlays tested have high
RFU and plastic plate sealers
were not effective at blocking
oxygen. An oxygen
impermeable, aluminum plate
sealer with minimal
autofluorescence or leaching of
aluminum was utilized.
A gain setting of 90 indicated
highest signal/background ratio.
Instrument parameters were
adjusted to recognize dimensions
specific to the Greiner Bio-One
Clear Bottom 96-well plate.
Linear regression data indicate
higher OCR in galactose
conditions. These media
conditions are consistent with
previous ATP assays associated
with this study.
Addition of probe to increase
probe/media ratios does not
enhance the signal seen due to
MX probe. Probe volume
should, however, remain
proportional to cell density.
Appropriate no-cell and no-probe
controls must be conducted per
experimental condition to be
subtracted during data analysis so
that OCR seen is due to probe.
Figure 6: Optimization of Oxygen Consumption Rate Assay Protocol
36
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.
37
r
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.
38
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.
39
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 1.6fold ± 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.
40
A)
B)
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.
41
A)
B)
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
42
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.
43
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.
44
A)
B)
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).
45
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.
46
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 proinflammatory 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
47
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 RBL2H3 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
48
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.
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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.
60