investigating effects of the antibiotic ciprofloxacin on the dopaminergic sh-sy5y cell line

71 Pages·2017·1.68 MB·English

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Masters Theses Dissertations and Theses
November 2015
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Jeffreys N. Johnson
University of Massachusetts Amherst
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Johnson, Jeffreys N., "INVESTIGATING EFFECTS OF THE ANTIBIOTIC CIPROFLOXACIN ON THE
DOPAMINERGIC SH-SY5Y CELL LINE" (2015). Masters Theses. 275.
https://doi.org/10.7275/7136809 https://scholarworks.umass.edu/masters_theses_2/275
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INVESTIGATING EFFECTS OF THE ANTIBIOTIC
CIPROFLOXACIN ON THE DOPAMINERGIC SH-SY5Y CELL
LINE
A Thesis Presented
by
JEFFREYS NATHANIEL JOHNSON
Submitted to the Graduate School of the
University of Massachusetts Amherst in partial fulfillment
of the requirements for the degree of
MASTER OF SCIENCE
September 2015
Neuroscience and Behavior
INVESTIGATING EFFECTS OF THE ANTIBIOTIC
CIPROFLOXACIN ON THE DOPAMINERGIC SH-SY5Y CELL
LINE
A Thesis Presented
by
JEFFREYS NATHANIEL JOHNSON
Approved as to style and content by:
James J. Chambers
Geng-Lin Li
Luke Remage-Healey
Melinda Novak, Department Chair
Neuroscience and Behavior
ABSTRACT
INVESTIGATING EFFECTS OF THE ANTIBIOTIC
CIPROFLOXACIN ON THE DOPAMINERGIC SH-SY5Y CELL
LINE
SEPTEMBER 2015
JEFFREYS NATHANIEL JOHNSON
B.A., CORNELL UNIVERSITY
M.S., UNIVERSITY OF MASSACHUSETTS AMHERST
Directed by: Professor James J. Chambers
Ciprofloxacin is a widely prescribed antibiotic which causes idiopathic sensory adverse
effects and is known to induce oxidative stress. Dopaminergic cells are known to have
intrinsic sensitivity to oxidative stress. To investigate whether ciprofloxacin potentiates cy-
totoxicity of dopamine, effects of combined drug treatments on cell viability were assessed by
resazurin reduction, and effects on mitochondrial health were assessed by morphology. The
cell viability assays suggest that ciprofloxacin significantly potentiates dopamine cytotoxicity
at clinically relevant doses, although dopamine possibly interferes with the viability assay.
Effects of drug treatments on mitochondrial morphology were inconclusive.
iii
TABLE OF CONTENTS
Page
ABSTRACT ............................................................... iii
LIST OF TABLES ......................................................... vi
LIST OF FIGURES ....................................................... viii
CHAPTER
1. INTRODUCTION ........................................................1
1.1 Ciprofloxacin...........................................................1
1.2 Mitochondria and oxidative stress.........................................2
1.3 Dopaminergic cells......................................................6
1.4 Current study: Motivations and hypothesis ................................7
2. METHODS ...............................................................9
2.1 Common methods ......................................................9
2.1.1 Cell culture......................................................9
2.1.2 Treatment conditions ............................................10
2.1.3 Viability assay ..................................................11
2.2 Effect of differentiation on SH-SY5Y sensitivity to dopamine ................12
2.3 Effect of ciprofloxacin on SH-SY5Y viability...............................12
2.4 Cell-free controls ......................................................13
2.5 Combined ciprofloxacin and dopamine treatment (2 ciprofloxacin doses).......13
2.6 Combined ciprofloxacin and dopamine treatment (8 ciprofloxacin doses).......14
2.7 Effects of ciprofloxacin and dopamine on mitochondrial morphology ..........15
3. RESULTS .............................................................. 17
3.1 Differentiated SH-SY5Y cells are sensitized to dopaminergic stress............17
iv
3.2 Ciprofloxacin, at physiologically relevant concentrations, is not acutely toxic
to SH-SY5Y cells ...................................................18
3.3 Dopamine reduces resazurin on its own ...................................19
3.4 Ciprofloxacin enhances toxicity of dopamine...............................19
3.5 Effects of ciprofloxacin and dopamine on mitochondrial morphology are
inconclusive........................................................23
4. DISCUSSION .......................................................... 35
4.1 Dopamine sensitivity dependence on cell differentiation .....................35
4.2 Viability dependence on ciprofloxacin.....................................36
4.3 Viability assay interference by dopamine ..................................37
4.4 Combined dopamine and ciprofloxacin treatment...........................40
4.5 Mitochondrial morphology ..............................................41
4.6 Future directions ......................................................43
APPENDIX: ADDITIONAL FIGURES.................................... 46
BIBLIOGRAPHY ......................................................... 53
v
LIST OF TABLES
Table Page
1.1 Reduction potentials. All half-reaction reduction potentials E and reaction
o?
potentials E are empirically determined in dilute solutions, pH 7-7.5,
h
relative to standard hydrogen electrode. A reduction/oxidation reaction
between two species is thermodynamically favored if the sum of
half-reaction potentials E is positive. So, for example, dopamine can
o?
reduce ubiquinone (Dopamine + Ubiquinone + 2e– + 2H+
Dopaminequinone + Ubiquinol + 2e– + 2H+,E =
h
E + 1 E = +206mV) but dopamine cannot reduce
o?,ubiquinone − ∗ o?,dopamine
NAD+ (E = E + 1 E = 159mV) ...................3
h o?,NAD+ − ∗ o?,dopamine −
2.1 Treatment conditions per plate, as ([Dopamine],[Cipro]) ....................14
2.2 Treatment conditions per plate, as ([Cipro],[Dopamine]) ....................15
3.1 p values for Figure 3.5..................................................21
3.2 Aspect ratio linear model parameters.....................................29
3.3 Form factor linear model parameters .....................................31
3.4 Area linear model parameters ...........................................33
3.5 Intensity linear model parameters........................................34
A.1 p values for Figure 3.3..................................................46
A.2 p values for Figure 3.2..................................................46
A.3 Number of cells analyzed per condition in Experiment 2 ....................47
vi
LIST OF FIGURES
Figure Page
1.1 Structure of ciprofloxacin. ...............................................1
1.2 A few reactions available to dopamine and its oxidation products.
Autooxidation of catechols into quinones by O typically proceeds via a
2
radical semiquinone intermediate. Quinones may participate in Michael
additions with nucleophilic protein sidechains or DNA bases to form
covalent adducts. Adapted from [46,69,86,105]. .........................2
3.1 (a) Curve fits and (b) dopamine IC50(viability) values for differentiated
(white) and undifferentiated (red) cells. p < 0.05 ......................17
∗
3.2 Effects of ciprofloxacin, alone, on cell viability. p < 0.0001 compared
∗∗∗∗
to untreated (black) cells.............................................18
3.3 Raw fluorescence values of drug-only controls for viability assay. See
Table A.1 for significance, compared to media (black). ...................20
3.4 (a) Curve fits and (b) dopamine IC50(viability) values for untreated (black)
and ciprofloxacin-treated (red) cells. p < 0.0001 ...................21
∗∗∗∗
3.5 Dopamine IC50(viability) dependence on ciprofloxacin. See Table 3.1 for
significance compared to untreated cells. ...............................22
3.6 Curve fits for dopamine IC50(viability) dependence on ciprofloxacin. See
Figure A.1 for curve fits plotted separately. ............................23
3.7 Representative images of MitoTracker Green fluorescence. Treatments are
shown as ([Cipro]/µg,[Dopamine]/µM). ...............................24
ml
3.8 Representative output of image processing pipeline, where individual
connected mitochondria objects are randomly colored. Treatments are
shown as ([Cipro]/µg,[Dopamine]/µM). ...............................25
ml
vii
3.9 Brightfield images of representative cells. Treatments are shown as
([Cipro]/µg,[Dopamine]/µM). ........................................26
ml
3.10 Empirical distribution functions of aspect ratio ............................27
3.11 Aspect ratio histograms, grouped by treatment ............................28
3.12 Empirical distribution functions of form factor.............................29
3.13 Form factor histograms, grouped by treatment.............................30
3.14 Empirical distribution functions of area...................................31
3.15 Area histograms, grouped by treatment...................................32
3.16 Empirical distribution functions of intensity ...............................33
3.17 Intensity histograms, grouped by treatment ...............................34
A.1 Curve fits for dopamine IC50(viability) dependence on ciprofloxacin, also
shown in Figure 3.6 .................................................48
A.2 (a) Curve fits and (b) dopamine IC50(viability) values for untreated (black)
and ascorbate-treated (red) cells. p < 0.0001 ......................49
∗∗∗∗
A.3 Normalized absorbance spectra of resazurin, without (blue) and with
(orange) 250µM dopamine. Oxidized dopamine absorbance reaches
half-maximal absorbance at 470 and overlaps with absorbance of media
at concentrations below 250µM (data not shown). ......................49
A.4 Transformed aspect ratio linear model residuals............................50
A.5 Transformed form factor linear model residuals ............................50
A.6 Transformed area linear model residuals ..................................50
A.7 Intensity linear model residuals ..........................................51
A.8 Linear mixed effect model ..............................................52
viii
CHAPTER 1
INTRODUCTION
1.1 Ciprofloxacin
HN
N N
OH
F
O O
Figure 1.1: Structure of ciprofloxacin.
Ciprofloxacin is a fluoroquinolone broad-spectrum bacteriocidal antibiotic used to treat
a wide variety of infections. It works by inhibiting bacterial DNA gyrase and topoisomerase
IV, which are required for DNA synthesis.
Historically, incidence of adverse effects had been estimated at about 13% of treated pa-
tients, with incidence of mild CNS-related adverse effects (headache and dizziness, resolving
within days) estimated at about 1% [10]. This has led to its prophylactic use, for example
in postal workers who are at risk of coming into contact with bioterror agents [13].
More recently, ciprofloxacin has been associated with acute psychiatric adverse reactions
(mania, psychosis, seizures) and with peripheral neuropathy [106]. Of the cases involving
neurological adverse effects, 42% reported disturbances in function of olfactory, visual, or
auditory senses [24,106]. In 2013, the FDA amended the label of ciprofloxacin to reflect the
“serious side effect of peripheral neuropathy, [which] may occur soon after these drugs are
taken and may be permanent [3].”
1