SELENIUM SUPPLEMENTATION
PROTECTS PLACENTAL TROPHOBLAST CELLS
FROM MITOCHONDRIAL OXIDATIVE STRESS
Alisha Khera M.Biotechnology
Submitted in fulfilment of the requirements of the degree of
Doctor of Philosophy
August 2015
School of Medical Science
Menzies Health Institute Queensland
Griffith University
ABSTRACT
Affecting 5-7% of all pregnancies, pre-eclampsia is a dangerous complication
of pregnancy which poses a serious health risk to both mother and fetus. It is a major
cause of maternal and perinatal morbidity and is characterised by hypertension,
proteinuria, edema and platelet aggregation. Despite the severity of the disease, no
comprehensive theory has been determined to explain the pathogenesis of the disease,
with delivery being the only curative treatment of this multisystem disorder of
pregnancy.
Oxidative stress plays a crucial role in the pathogenesis of pre-eclampsia,
which results in placental injury causing a hypoxic environment and eventually levels
of anti-oxidants are markedly decreased. This leads to the activation of cell death
pathways such as apoptosis and necrosis.
The findings reported in this thesis suggest that selenium supplementation
could be a potential therapy for treating women who are at risk of developing preeclampsia. In this study, selenium supplementation was used to up-regulate the
expression of endogenous anti-oxidants, Glutathione Peroxidase (GPx) and
Thioredoxin-Reductase (Thx-Red). It has been well documented in the literature that
pre-eclampsia is caused by oxidative stress and in an attempt to address this we
investigated whether oxidative stress in trophoblast-like cells is protected by selenium
supplementation. Trophoblast-like cells, BeWo, JEG-3 and Swan-71 cells were
exposed to endogenous stressors such as Rotenone and Antimycin which are able to
generate mitochondrial oxidative stress by inhibiting the electron transport chain.
ii
Incubation with selenium was found to protect trophoblast cells from oxidative stress
by reducing the generation of Reactive Oxygen Species (ROS).
Placental apoptosis plays a significant role in the normal development of
placenta. However, apoptosis is also involved in the pathophysiology of pre-eclampsia,
by activating the death receptor pathways through the intrinsic or mitochondrial
pathway. It was the aim of this thesis to induce the apoptosis by increasing
mitochondrial ROS production with Rotenone and thereby protecting the cells from
undergoing apoptosis by supplementation with selenium. Western blot analysis
revealed that there was a significant down-regulation in the expression of Bcl-2
apoptotic marker when cells were given Rotenone treatment. However, when selenium
supplementation was given in combination with Rotenone, it not only up-regulated the
expression of Bcl-2 but also improved the viability of trophoblast cells. This suggests
that selenium supplementation is able to protect trophoblast cells from apoptosis
through GPx and Thx-Red up-regulation.
Importantly the work presented in this thesis shows that selenium treatment
enhances the mitochondrial function of trophoblast cells and also activates
mitochondrial biogenesis through selenoprotein H (Sel H) and transcription factors
such as Nuclear Respiratory Factor-1 (NRF-1) and Peroxisome Proliferator-Activated
Receptor Coactivator-1alpha (PGC-1α). Selenium supplementation significantly
boosted mitochondrial respiration in trophoblast cells when measured by Oxygraph.
Quantitative real-time PCR data revealed that there was higher mitochondrial content
in trophoblast cells when they were given selenium treatment. Expression of
mitochondrial biogenesis proteins Sel H, PGC-1α and NRF-1 examined by western
blotting was also significantly higher with selenium supplementation in BeWo, JEG-3
iii
and Swan-71 cells. The analysis of these results suggests that selenium is responsible
for mitochondrial regeneration in addition to protecting cells from oxidative stress. To
validate our findings, the effect of selenium was studied on first trimester placental
tissues which were supplemented with selenium at different time points. Mitochondrial
respiration was significantly enhanced by selenium treatment at an early time point.
Quantitative real-time PCR analysis of placental samples revealed that mRNA
expression of GPx and Thx-Red was enhanced with selenium supplementation.
Expression of Sel H, PGC-1α and NRF-1 was also increased with selenium
supplementation. These results indicate that protective effect of selenium was observed
not only in BeWo, JEG-3 and Swan-71 cells but also in first trimester placental tissues.
In summary, the data obtained in thesis clearly suggest that selenium
supplementation contributes to the reduction of placental oxidative stress and upregulates the activity of anti-oxidants such as GPx and Thx-Red. The novel data
obtained from this study will provide the better understanding of pathophysiology of
pre-eclampsia. The understanding gained from this study provides a new insight that
selenium supplementation could be a cost-effective and readily available method
which could help in reducing the impact of pre-eclampsia.
iv
STATEMENT OF ORIGINALITY
This work has not been previously submitted for a degree or diploma to any
university. To the best of my knowledge and belief, the thesis contains no material
previously published or written by another person except where due reference is made
in the thesis itself.
____________________
Alisha Khera
August 2015
v
ABSTRACT
TABLE OF CONTENTS
.............................................................................................................. ii
STATEMENT OF ORIGINALITY ............................................................................... v
LIST OF FIGURES ..................................................................................................... xvi
LIST OF TABLES..................................................................................................... xxiii
ACKNOWLEDGEMENTS...................................................................................... xxiiii
LIST OF PUBLICATIONS ....................................................................................... xxiv
CONFERENCE PRESENTATIONS: ........................................................................ xxv
LIST OF ABBREVIATIONS .................................................................................. xxvii
CHAPTER 1 INTRODUCTION ................................................................................. 1
1.1
DEFINITION AND DIAGNOSIS OF PRE-ECLAMPSIA ................. 2
1.2
EPIDEMIOLOGY OF PRE-ECLAMPSIA.......................................... 3
1.3
RISK FACTORS AND TREATMENT OF PRE-ECLAMPSIA ......... 3
1.4
PLACENTA AND PRE-ECLAMPSIA ............................................... 5
1.5
PLACENTAL ANATOMY ................................................................. 7
1.6
AETIOLOGY OF PRE-ECLAMPSIA ................................................. 8
1.7
PLACENTAL OXIDATIVE STRESS AND APOPTOSIS ............... 13
1.8
ANTI-OXIDANTS ............................................................................. 15
vi
1.9
MECHANISM OF ACTION OF ENDOGENOUS ANTI-OXIDANTS
.......................................................................................................... 16
1.10 ROLE OF ANTI-OXIDANTS IN PREGNANCY AND PREECLAMPSIA...................................................................................... 18
1.11 SELENIUM (SE) AND SELENOPROTEINS................................... 19
1.12 PRE-ECLAMPSIA AND SELENIUM .............................................. 20
1.13 IN-VITRO MODELS OF TROPHOBLAST CELLS ......................... 23
1.14 SUMMARY AND RESEARCH PLAN............................................. 24
CHAPTER 2 GENERAL METHODS ..................................................................... 26
2.1
CELL CULTURE ............................................................................... 27
2.1.1 General cell culture .......................................................................... 27
2.1.2 Cell Preparations .............................................................................. 28
2.1.3 Cell Extraction.................................................................................. 28
2.2
BIOCHEMICAL METHODS ............................................................ 28
2.2.1 Protein Estimations .......................................................................... 28
2.2.2 Anti-oxidant Assays ......................................................................... 29
2.2.2.1 Glutathione Peroxidase Assay .......................................................... 29
2.2.2.2 Thioredoxin-Reductase Assay.......................................................... 31
vii
2.3 ASSAYS FOR MEASURING CELL VIABILITY ............................... 32
2.3.1 3-[4, 5-dimethylthiazol-2-yl]-2, 5 diphenyl tetrazolium bromide
(MTT) Assay .................................................................................... 32
2.3.2 Resazurin End Point Assay .............................................................. 34
2.4
ASSAYS FOR MEASURING OXIDATIVE STRESS ..................... 35
2.4.1 2’,7’-Dichlorofluorescein diacetate (DCFDA) Assay ...................... 35
2.5
REAL TIME POLYMERASE CHAIN REACTION (PCR) ............. 37
2.6
cDNA SYNTHESIS ........................................................................... 38
2.7
WESTERN BLOTTING .................................................................... 38
2.8
STATISTICAL ANALYSIS .............................................................. 40
2.8.1 One-way Analysis of Variance ........................................................ 40
2.8.2 Two-tailed test .................................................................................. 41
CHAPTER 3 EXPRESSION OF GPx AND Thx-Red IN RESPONSE TO
SELENIUM SUPPLEMENTATION ............................................. 42
3.1
ABSTRACT ....................................................................................... 44
3.2
INTRODUCTION .............................................................................. 45
3.3
AIMS .................................................................................................. 47
3.4
MATERIALS AND METHODS ....................................................... 48
3.4.1 Cell Culture plus Selenium supplementations ................................. 48
viii
3.4.2 Cell Extracts and Biochemical Analysis .......................................... 48
3.4.3 Protein Estimations .......................................................................... 49
3.4.4 Glutathione Peroxidase Assay .......................................................... 49
3.4.5 Thioredoxin-Reductase Assay.......................................................... 49
3.4.6 Western Blotting .............................................................................. 50
3.4.7 Statistical Analysis ........................................................................... 50
3.5
RESULTS ........................................................................................... 51
3.5.1 Expression of GPx protein in response to selenium supplementation
.......................................................................................................... 51
3.5.2 Baseline activity of GPx in BeWo, JEG-3 and Swan-71 cell lines .. 52
3.5.3 Dose dependent expression of GPx in Swan-71 in response to
selenium ........................................................................................... 52
3.5.4 Expression
of
Thx-Red
protein
in
response
to
selenium
supplementation ............................................................................... 53
3.5.5 Baseline Activity of Thx-Red in BeWo, JEG-3 and Swan-71 cell
lines .................................................................................................. 54
3.5.6 Dose dependent Activity of Thx-Red in Swan-71 cell lines
supplemented with increasing concentrations of selenium .............. 55
3.5.7 Effect of NaSe and SeMet toxicity on BeWo, JEG-3 and Swan-71
cells via MTT ................................................................................... 56
3.5.8 Effect of NaSe and SeMet toxicity on BeWo, JEG-3 and Swan-71
cells via Resazurin ............................................................................ 58
ix
3.5.9 Up-regulation of GPx in BeWo, JEG-3 and Swan-71 cell lines in
response to selenium ........................................................................ 60
3.5.10 Up-regulation of Thx-Red in BeWo, JEG-3 and Swan-71 cell lines in
response to selenium ........................................................................ 61
3.6
DISCUSSION ..................................................................................... 63
CHAPTER 4 SELENIUM AND MITOCHONDRIAL OXIDATIVE STRESS ... 67
4.1
ABSTRACT ....................................................................................... 69
4.2
INTRODUCTION .............................................................................. 70
4.3
AIMS .................................................................................................. 72
4.4
MATERIALS AND METHODS ....................................................... 73
4.4.1 Cell Culture and Treatments ............................................................ 73
4.4.2 Quantification of ROS levels and selenium supplementation .......... 73
4.4.3 MTT and Resazurin end point assay for cell viability ..................... 74
4.4.4 Statistical Analysis ........................................................................... 74
4.5
RESULTS ........................................................................................... 75
4.5.1 ROS Production in Trophoblast cells post Rotenone and Antimycin
treatment ........................................................................................... 75
4.5.2 Selenium supplementation reduces ROS production induced by
Rotenone and Antimycin .................................................................. 76
x
4.5.3 Cellular viability measured by MTT in Swan-71 cells when treated
with Rotenone and effect of NaSe supplementation ........................ 77
4.5.4 Cellular viability measured by MTT in Swan-71 cells when treated
with Antimycin and effect of NaSe supplementation ...................... 78
4.5.5 Cellular activity in BeWo, JEG-3, Swan-71 when oxidatively
stressed with Rotenone as assessed with Reszaurin ......................... 80
4.5.6 Cellular activity of Antimycin in BeWo, JEG-3 and Swan-71 cells
assessed via Resazurin ..................................................................... 81
4.5.7 Sodium Selenite (NaSe) protection from oxidative stress induced by
Rotenone........................................................................................... 82
4.5.8 Selenomethionine (SeMet) protection from oxidative stress induced
by Rotenone...................................................................................... 84
4.5.9 Sodium Selenite (NaSe) protection from oxidative stress induced by
Antimycin ......................................................................................... 86
4.5.10 Selenomethionine (SeMet) protection from oxidative stress induced
by Antimycin .................................................................................... 88
4.6
DISCUSSION ..................................................................................... 90
CHAPTER 5 SELENIUM SUPPLEMENTATATION AND PROTECTION
FROM APOPTOSIS ........................................................................ 95
5.1
ABSTRACT ....................................................................................... 96
xi
5.2
INTRODUCTION .............................................................................. 97
5.3
AIMS .................................................................................................. 98
5.4
MATERIALS AND METHODS ....................................................... 99
5.4.1 Cell Culture ...................................................................................... 99
5.4.2 Selenium Treatment ......................................................................... 99
5.4.3 Rotenone Treatment ......................................................................... 99
5.4.4 Western Blotting .............................................................................. 99
5.4.5 Flow Cytometry.............................................................................. 100
5.4.6 Statistical Analysis ......................................................................... 100
5.5
RESULTS ......................................................................................... 102
5.5.1 Expression of Bcl-2 induced by Rotenone and effect of NaSe
supplementation on apoptosis in trophoblast cells ......................... 102
5.5.2 Quantification of apoptosis in trophoblast cells using Annexin V
FITC ............................................................................................... 104
5.6
DISCUSSION ................................................................................... 106
CHAPTER 6 SELENIUM INDUCED MITOCHONDRIAL BIOGENESIS IN
TROPHOBLAST ............................................................................ 109
6.1
ABSTRACT ..................................................................................... 111
6.2
INTRODUCTION ............................................................................ 112
xii
6.3
AIMS ................................................................................................ 114
6.4
MATERIALS AND METHODS ..................................................... 115
6.4.1 Cell Culture .................................................................................... 115
6.4.2 Measurement of mitochondrial respiration in trophoblast –like cell
lines ................................................................................................ 115
6.4.3 DNA Extraction.............................................................................. 116
6.4.4 Real time Polymerase Chain Reaction (RT-qPCR) for determining
mitochondrial DNA copy number .................................................. 117
6.4.5 Western Blotting ............................................................................ 118
6.4.6 Citrate Synthase Assay ................................................................... 118
6.4.7 Statistical Analysis ......................................................................... 119
6.5
RESULTS ......................................................................................... 120
6.5.1 Selenium enhances mitochondrial respiration in trophoblast -like cell
lines ................................................................................................ 120
6.5.2 Selenium increases mitochondrial content in trophoblast-like cells
........................................................................................................ 122
6.5.3 Selenium up-regulates mitochondrial biogenesis markers ............. 124
6.6
CHAPTER
DISCUSSION ................................................................................... 128
7
SELENIUM
SUPPLEMENTATION
PROMOTES
MITOCHONDRIAL FUNCTION IN PLACENTAL TISSUES .....
.......................................................................................................... 132
xiii
7.1
ABSTRACT ..................................................................................... 133
7.2
INTRODUCTION ............................................................................ 134
7.3
AIMS ................................................................................................ 136
7.4
MATERIALS AND METHODS ..................................................... 137
7.4.1 Tissue collection and culture .......................................................... 137
7.4.2 Western Blotting ............................................................................ 138
7.4.3 DNA, RNA and protein extractions ............................................... 139
7.4.4 cDNA synthesis .............................................................................. 139
7.4.5 Real time Polymerase Chain Reaction (RT-qPCR) for determining
the expression of anti-oxidant enzymes ......................................... 140
7.4.6 Real time Polymerase Chain Reaction (RT-qPCR) for determining
mitochondrial DNA copy number. ................................................. 141
7.4.7 Measurement of mitochondrial respiration .................................... 142
7.4.8 Statistical Analysis ......................................................................... 143
7.5
RESULTS ......................................................................................... 144
7.5.1 Expression of GPx and Thx-Red anti-oxidant enzymes in first
trimester placental tissues............................................................... 144
7.5.2 GPx and Thx-Red anti-oxidant enzymes in term placental tissues 145
7.5.3 mRNA expression of GPx and Thx-Red in first trimester placental
tissues ............................................................................................. 146
xiv
7.5.4 Selenium upregulates mitochondrial biogenesis markers in first
trimester placental tissues............................................................... 147
7.5.5 Selenium may increase mitochondrial content in first trimester
placental tissues .............................................................................. 151
7.5.6 Selenium increases mitochondrial respiration in first trimester
placental tissues .............................................................................. 152
7.6
DISCUSSION ................................................................................... 154
CHAPTER 8 GENERAL DISCUSSION ............................................................... 158
REFERENCES ......................................................................................................... 170
xv
LIST OF FIGURES
CHAPTER 1
Figure 1.1 Trophoblast invasion and uterine spiral artery remodelling in normal
pregnancy and pre-eclamptic pregnancy (Wang et al., 2009). .................... 6
Figure 1.2 Structure of human placenta (Sood et al., 2006). .......................................... 8
Figure 1.3 Production of ROS through mitochondrial electron transport chain
(Hamanaka et al., 2010). ............................................................................................... 10
Figure 1.4 Trophoblast cells inducing apoptosis by the secretion of TNF-α and
Indolamine 2, 3-dioxygenase (IDO) (Kaufmann et al., 2003). ............... 12
Figure 1.5 Morphological features of apoptosis (Microbiology Bytes: Virology, 2007).
...................................................................................................................................... 14
Figure 1.6 Schematic summary of Glutathione-associated anti-oxidant systems
(Nordberg et al., 2001). ........................................................................... 17
Figure 1.7 Enzymatic reactions of Thioredoxin System (Nordberg et al., 2001). ....... 18
CHAPTER 2
Figure 2.1 MTT is converted into Formazan (Ebada et al., 2008) ............................... 33
Figure 2.2 Schematic representation of reactions involved in 2’, 7’ Dichlorofluorescein
diacetate (DCFDA) Assay (Hensley et al., 2003). .................................... 36
CHAPTER 3
Figure 3.1 Expression of GPx protein in Swan-71cells when supplemented with
increasing amounts of NaSe (A). Each blot was repeated 4 times and a
representative blot is shown in panel A. Densitometry and statistical
analysis is shown in panel B ...................................................................... 51
Figure 3.2 Baseline activity of GPx in in BeWo, JEG-3 and Swan-71cell lines.......... 52
xvi
Figure 3.3 Dose dependent activity of GPx in Swan-71, when supplemented with NaSe
(A) or SeMet (B) for 24 h treated with increasing concentrations of
selenium. .................................................................................................... 53
Figure 3.4 Expression of Thx-Red protein in Swan-71 cells when supplemented with
increasing amounts of NaSe. Each blot was repeated 4 times and a
representative blot is shown in panel A. Densitometry and statistical analysis
is shown in panel B as mean ± standard deviation (S.D). ............................ 54
Figure 3.5 Baseline activity of Thx-Red in BeWo, JEG-3 cell lines and Swan-71 cells.
...................................................................................................................................... 55
Figure 3.6 Dose dependent activity of Thx-Red in Swan-71, when supplemented with
NaSe (A) or SeMet (B) for 24 h treated with increasing concentrations of
selenium. ...................................................................................................... 56
Figure 3.7 Effect of NaSe (blue bars) and SeMet (violet bars) on cellular viability and
toxicity measured by MTT on BeWo, JEG-3 and Swan-71 cells. ............... 58
Figure 3.8 Effect of NaSe (blue bars) and SeMet (violet bars) on cellular viability and
toxicity measured by Resazurin on BeWo, JEG-3 and Swan-71 cells. ....... 59
Figure 3.9 Up-regulation of GPx activity in in BeWo (A), JEG-3 (B) and Swan-71cell
lines (C) when supplemented with 100nM NaSe and 500nM SeMet. ......... 61
Figure 3.10 Up-regulation of GPx activity in in BeWo (A) and Swan-71cell lines (B)
when supplemented with 100nM NaSe and 500nM SeMet. ..................... 62
CHAPTER 4
Figure 4.1 Effect of Antimycin (A) and Rotenone (B) on ROS production as compared
to control in Swan-71 cells. ....................................................................... 75
xvii
Figure 4.2 Effect of NaSe supplementation and SeMet treatment on ROS production
with Antimycin (A, B) and Rotenone (C, D) treatment for 4 hours. ........... 76
Figure 4.3 Effect of Rotenone cellular viability on Swan-71 cells measured by MTT
assay in addition with 100nM NaSe supplementation. ................................ 78
Figure 4.4 Effect of Antimycin on cellular viability of Swan-71 cells measured by
MTT assay in addition with 100nM NaSe supplementation. .................... 79
Figure 4.5 Effect of Rotenone on cellular activity of BeWo (A), JEG-3 (B) and Swan71 (C) cell lines respectively. ....................................................................... 80
Figure 4.6 Effect of Antimycin on cellular activity of BeWo (A), JEG-3 (B) and Swan71(C) cell lines respectively when they are oxidatively stressed. ................ 82
Figure 4.7 Effect of Rotenone on percentage cellular activity in BeWo (A), JEG-3 (B)
and Swan-71 cells (C), treated with NaSe for 24 hours and subsequently
treated with increasing doses of Rotenone concentration. ........................... 83
Figure 4.8 Effect of Rotenone on percentage cellular activity in BeWo (A) JEG-3 (B)
and Swan-71 cells (C), treated with SeMet for 24 h and later treated with
increasing doses of Rotenone concentration. ............................................... 85
Figure 4.9 Effect of Antimycin on percentage cellular activity in BeWo (A), JEG-3 (B)
and Swan-71 cells (C), treated with NaSe for 24 hours and subsequently
treated with increasing doses of Antimycin concentration. ......................... 87
Figure 4.10 Effect of Antimycin on percentage cellular activity in BeWo (A) JEG-3
(B) and Swan-71 cells (C), treated with SeMet for 24 h and later treated
with increasing doses of Antimycin concentration. ................................... 89
xviii
CHAPTER 5
Figure 5.1 Bcl-2 expression in BeWo (A), JEG-3 (B) and Swan-71 (C) cells treated
with selenium and later exposed to Rotenone. ........................................ 102
Figure 5.2 Densitometry of western blots quantitating the expression levels of Bcl-2 in
BeWo (A), JEG-3 (B) and Swan-71 (C) cells normalised to actin
expression................................................................................................... 103
Figure 5.3 Annexin V FITC staining in untreated BeWo cells (A), when cells are
exposed to 100μM Rotenone (B) and cells treated in combination with
selenium and Rotenone (C). Annexin V FITC staining in untreated JEG-3
cells (D), when cells are exposed to 100μM Rotenone (E) and cells treated in
combination with selenium and Rotenone (F). .......................................... 105
CHAPTER 6
Figure 6.1 (A) Intact cells; a representative oxygraph trace of oxygen flux relative to
cell number in non-selenium (light grey) and selenium-treated (dark grey)
cells. Mitochondrial ROUTINE and ETS respiration rates observed in
BeWo, JEG-3 and Swan-71 cells, respectively. (B) Permeabilized Swan-71
cells. ......................................................................................................... 121
Figure 6.2 (A) Mitochondrial content is increased in Swan-71 cells lines Supplemented
with selenium. (A) Mitochondrial DNA copy number (mtDNA/nDNA
ratio), in Swan-71 cells treated with 100nM NaSe compared to untreated
controls and (B) 500nM SeMet compared to untreated controls. (C) Citrate
synthase activity in Swan-71 cells treated with 100nM NaSe compared to
untreated controls..................................................................................... 123
xix
Figure 6.3 Sel H expression in BeWo, JEG-3 and Swan-71 cells treated with selenium
100nM NaSe (A) and 500nM SeMet (B) compared to controls. . ............. 125
Figure 6.4 PGC-1 α expression in Swan-71 cells treated with selenium 100nM NaSe
(A) and 500nM SeMet (B) compared to controls. Images show
representative western blot bands. NRF-1 expression in Swan-71 cells with
selenium 100nM NaSe (C) and 500nM SeMet (D) compared to controls..126
CHAPTER 7
Figure 7.1 GPx and Thx-Red expression in 1st trimester placental tissues treated with
100nM NaSe for 4 and 12 hours compared to controls. ............................ 144
Figure 7.2 GPx and Thx-Red expression in term placental tissues immediately after
collection (Time 0) and after treatment with 100nM NaSe for 4 hours
compared to controls. ................................................................................. 145
Figure 7.3 GPx and Thx-Red mRNA expression in placental tissues treated with
100nM NaSe for 4 and 12 hours compared to controls. .......................... 147
Figure 7.4 Sel H expression in placental tissues treated with 100nM NaSe for 4, 12, 24,
48 and 96 hours compared to controls. ...................................................... 148
Figure 7.5 PGC-1α expression in placental tissues treated with 100nM NaSe for 4, 12,
24, 48 and 96 hours compared to controls. ................................................ 149
Figure 7.6 NRF-1 expression in placental tissues treated with 100nM NaSe for 4, 12,
24, 48 and 96 hours compared to controls. ................................................ 150
Figure 7.7 Mitochondrial content measured in placental tissues when supplemented
with selenium for 4, 12, 24, 48 and 96 hours. ............................................ 152
xx
Figure 7.8 Effect of Selenium treatment on oxidative phosphorylation through
complexes IV, II and I, and LEAK respiration in first trimester placental
tissues. ...................................................................................................... 153
xxi
LIST OF TABLES
CHAPTER 6
Table 6.1 Real time PCR primers ................................................................... 118
CHAPTER 7
Table 7.1 Real time PCR primers ................................................................... 140
Table 7.2 Real time PCR primers ................................................................... 141
xxii
ACKNOWLEDGEMENTS
First and foremost, I would like to thank my supervisors Prof Tony Perkins and
Dr Jessica Vanderlelie for providing me the opportunity to carry out PhD under their
excellent supervision. Tony, I thank you for sharing your knowledge and passion for
research with me and belief that I would be successful in my results. Without Prof
Perkins I could not have made though my PhD and for this I’m sincerely grateful.
Second big thanks to Dr Jessica Vanderlelie who has always been very helpful.
She has always been supportive throughout my thesis and encouraged me.
I also thank Dr Olivia Holland for giving me an opportunity to work with
placental tissues.
Very special thanks to Dr Jelena Vider for teaching me such an excellent skill
of flow cytometry. Lena, you have always supported me both personally and
professionally, especially the support I have got from you in last few months of my
PhD was incredible and tremendously appreciative.
Thanks must also go to my past and present friends of Griffith University:
Rebecca Greally, Javed Fowder and Jeetu for always supporting me in my hard times
and being “there”. Without their friendship, advice and encouragement my journey of
PhD would have been difficult.
Finally, I would like to thank my family especially my mum, dad and brother
for their constant support, encouragement and having faith in me. I remember God for
making me stand through the difficult times when I had almost given up.
xxiii
LIST OF PUBLICATIONS
Publications arising from the work presented in this thesis:
Khera A, Vanderlelie JJ, Holland OJ, Perkins AV. (2015) Expression of Glutathione
Peroxidase and Thioredoxin-Reductase in response to selenium supplementation.
Journal of Trace Elements in Medicine and Biology: (Manuscript in preparation).
*Data for this manuscript was derived from Chapter 3 of this thesis
Khera A, Vanderlelie JJ, Perkins AV. (2013) Selenium supplementation protects
trophoblast cells from mitochondrial oxidative stress. Placenta 34: 594-8.
*Data for this manuscript was derived from Chapter 4 of this thesis
Khera A, Dong LF, Holland O, Vanderlelie J, Pasdar EA, Neuzil J, Perkins AV.
(2015) Selenium supplementation induces mitochondrial biogenesis in trophoblasts.
Placenta 36: 863-9.
*Data for this manuscript was derived from Chapter 6 of this thesis
xxiv
CONFERENCE PRESENTATIONS:
Khera A, Vanderlelie JJ, Perkins AV. (2012) Selenium supplementation protects
trophoblast cells from mitochondrial oxidative stress. Presented at the Endocrine
Society of Australia and Society for Reproductive Biology, Gold Coast, Australia.
Khera A, Vanderlelie JJ, Perkins AV. (2012) Selenium supplementation protects
trophoblast cells from mitochondrial oxidative stress. Presented at the Gold Coast
Medical Research Conference, Gold Coast, Australia.
Khera A, Vanderlelie JJ, Perkins AV. (2013) Selenium supplementation protects
trophoblast cells from mitochondrial oxidative stress. Presented at the Endocrine
Society of Australia and Society for Reproductive Biology, Sydney, Australia.
Khera A, Vanderlelie JJ, Perkins AV. (2013) Selenium supplementation protects
trophoblast cells from mitochondrial oxidative stress. Presented at the Gold Coast
Medical Research Conference, Gold Coast, Australia.
Khera A, Vanderlelie JJ, Holland OJ, Perkins AV. (2014) Selenium supplementation
increases trophoblast mitochondrial content. Presented at the Gold Coast Medical
Research Conference, Gold Coast, Australia.
Khera A, Holland OJ, Vanderlelie JJ, Perkins AV. (2015) Trophoblast mitochondrial
biogenesis and functionality is increased with selenium supplementation. Presenting at
the International Federation of Placental Associations, Brisbane, Australia.
xxv
LIST OF ABBREVIATIONS
%
Percentage
°C
Degree Celsius
±
Plus or minus
>
Greater than
Α
Alpha
Β
Beta
Γ
Gamma
ADP
Adenosine diphosphate
ANOVA
Analysis of variance
ATCC
American Tissue Culture
Collection
ATP
Adenosine 5’-triphosphate
BB
Blood Pressure
BCA
B-cell lymphoma 2
BSA
Bovine serum albumin
CASPASES
Cysteine-aspartate-specific
proteases
Cc
Centimetre Square (cm2)
CO2
Carbon Dioxide
CI
Complex I
CII
Complex II
CIII
Complex III
CIV
Complex IV
Cdna
Complementary DNA
xxvi
Ct
The difference in Ct
values
Ct
Cycle Threshold
DCF
2’, 7’-Dichlrofluorescin
DCFDA
2’, 7’-Dichlrofluorescin
diacetate Assay
DMEM
Dulbecco’s modified
Eagle’s medium
DMSO
Dimethyl sulfoxide
DNA
Deoxyribonucleic acid
DPBS
Dulbecco’s Phosphate
Buffered Saline
DTNP
5, 5’- dithio-bis (2dinitrobenzoic acid)
ECL
Enhanced
chemiluminescence
EDTA
Ethylenediaminetetraacetic
acid
EGTA
Ethylene glycol tetraacetic
acid
ETC
Electron Transport Chain
ETS
Electron Transfer System
FCCP
carbonyl cyanide ptrifluoro-methoxyphenyl
hydrazone
FITC
Fluorescein isothiocyanate
G
Relative centrifugal force
GPX
Glutathione Peroxidase
GR
Glutathione Reductase
xxvii
GSH
Glutathione
GSSG
Oxidised Glutathione
H
Hour
H2O2
Hydrogen Peroxide
HCG
Human chorionic
gonadotropin
Hg
Millimetre of Mercury
HRP
Streptavidin-horseradish
peroxidase
Htert
Human Telomerase
Reverse Transcriptase
IDO
Indolamine 2, 3dioxygenase
IgG
Immunoglobulin
IL
Interleukin
IUGR
Intrauterine Growth
Restriction
Kpi
Potassium phosphate
KH2PO4
Monopotassium phosphate
M
Molar
µg
Microgram
Mg
Milligram
mg/mL
Milligram per millilitre
µL
Microliter
μM
Micromolar
mM
Millimolar
MgCl2
Magnesium chloride
xxviii
Min
Minute
Mn-SOD
Manganese superoxide
dismutase
MiRO
Mitochondrial Respiration
Medium
mtDNA
Mitochondrial
Deoxyribonucleic acid
mU
Milliunits
mRNA
Messenger RNA
MTT
3-[4, 5-dimethylthiazol-2yl]-2, 5 diphenyl
tetrazolium bromide
NaCl
Sodium Chloride
NADH
Nicotinamide Adenine
nucleotide Phosphate
nDNA
Nuclear Deoxyribonucleic
acid
NaSe
Sodium Selenite
Ng
Nerve Growth Factor
NGF
Nanomolar
nM
Nanometre
NRF-1
Nuclear Respiratory
Factor-1
NTC
Non-template controls
O2 -
Super oxide Anion
OXPHOS
Oxidative Phosphorylation
PBS
Phosphate Buffered Saline
PCR
Polymerase Chain
Reaction
xxix
PGC-1α
Peroxisome ProliferatorActivated Receptor
Coactivator-1alpha
PI
Propidium Iodide
PMSF
Phenylmethylsulfonyl
fluoride
PVDF
Polyvinylidene fluoride
qRT-PCR
Quantitative real-time
PCR
U
Unit
UDG
Selenocysteine, Aspartic
acid and Glycine
U/ml
Units per millilitre
RNA
Ribonucleic acid
RONS
Reactive oxygen and
nitrogen species
ROS
Reactive Oxygen Species
ROX
Residual Oxygen
Consumption
RPM
Revolutions per minute
SD
Standard Deviation
SD-PAGE
Sodium dodecyl sulfate
polyacrylamide gel
electrophoresis
Se
Selenium
SeMet
Selenomethionine
Sel H
Selenoprotein H
-SH
Sulfhydryl
SOD
Superoxide dismutase
xxx
TBS
Tris buffered saline
TBS-T
Tris buffered salineTween
Thx
Thioredoxin
Thx-Red
Thioredoxin-Reductase
Thx-S2
Oxidised Thioredoxin
TMPD
N,N,N',N'-Tetramethyl-pphenylenediamine
dihydrochloride
TNB
5-thio-2-nitrobenzoic acid
TNF-α
Tumor necrosis factor-α
TPx
Thioredoxin Peroxidase
Tris-Hcl
Tris-Hydrochloride
xxxi
CHAPTER 1
GENERAL INTRODUCTION
General Introduction
1.1
Chapter 1
DEFINITION AND DIAGNOSIS OF PRE-ECLAMPSIA
Pre-eclampsia is defined as an abnormal condition of pregnancy, which is
clinically diagnosed by hypertension, proteinuria, edema and platelet aggregation after
20 weeks of gestation Pre-eclampsia occurs in 5-7% of pregnancies and is a major
cause of maternal mortality in developed and developing countries (Sibai et al., 2005).
There is no cure for the disease except the early delivery of the foetus and placenta.
According to the Society of Obstetric Medicine of Australia and New Zealand,
2014 the guidelines of the clinical diagnosis of pre-eclampsia includes hypertension,
which is one of the main characteristic features with a systolic Blood pressure of 160170mm Hg and diastolic BP of 110mmHg or higher (Lowe et al., 2015). Peripheral
edema is also a comorbidity often associated with the development of pre-eclampsia.
Secondary symptoms such as epigastric pain, low platelet count and neurological
effects may signify progression to HELLP (hemolysis, elevated liver enzymes, low
platelets) syndrome; a life threatening condition characterised by multisystem organ
failure and seizures. Management of pre-eclampsia is coordinated to maximise fetal
time in utero whilst ensure maternal symptoms do not deteriorate with the presence of
HELLP indicators a clear indication to deliver the baby and remove the placenta from
the uterus (VanWijk et al., 2000).
Pre-eclampsia is considered to be a disease of placental origin and occurs
following shallow trophoblast invasion and subsequent poor placentation. Oxidative
stress plays a critical role in the pathogenesis of pre-eclampsia and there have been
many studies demonstrating an imbalance between Reactive Oxygen Species (ROS)
and anti-oxidants in placental tissues (Hubel, 1999, Redman et al., 2000). Trophoblast
cells within the placenta are susceptible to oxidative stress and the increased ROS
2
General Introduction
Chapter 1
generated in pre-eclampsia trigger higher levels of trophblastic cell turnover, and
release of placental debris into the maternal circulation (Cindrova, 2009). This debris
may be apoptotic, necrotic and mitophagic in nature and causes activation of a
systemic immune system and a cascade of pro-inflammatory events resulting in
endothelial cell dysfunction and clinical manfiestations of pre-eclampsia (Redman et
al., 2001). Hence, understanding the link between oxidative stress and placental cell
turnover will potentially lead to improved therapies for treating this disease.
1.2
EPIDEMIOLOGY OF PRE-ECLAMPSIA
Pre-eclampsia affects between 2 and 7% of all pregnancies in the Western
world (Villar et al., 2004). The World Health Organization (2005) estimates about
12% of maternal deaths worldwide are associated with pre-eclampsia, which places a
considerable burden on health care resources globally (World Health Report, 2005). In
the context of the developing world up to up to 42% of maternal deaths attributable
may be attributable to pre-eclampsia, equating to approximately 60,000 mothers per
year (Noris et al., 2005).
1.3
RISK FACTORS AND TREATMENT OF PRE-ECLAMPSIA
A number of risk factors are associated with pre-eclampsia. Women who are at
high risk of pre-eclampsia are diagnosed before 13 week of gestation. High risk
women include those women who previously have hypertension, chronic kidney
disease and previous early onset of pre-eclampsia. The risk of pre-eclampsia is higher
at age more than 40 years, women who become pregnant with donor eggs, embryo
implantation, have family history of pre-eclampsia and women who are suffering from
3
General Introduction
Chapter 1
medical conditions antiphospholipid syndrome (English et al., 2015). Other risk risk
factors for pre-eclampsia are insulin resistance with obesity and diabetes, which are
also related with an increased incidence of pre-eclampsia (Walker, 2000 and Wolf et
al., 2002). In developing countries, malnutrition has been identified as an important
risk factor (Noris et al., 2005). Some genetic factors are also involved in the
development of pre-eclampsia. There have been reports which suggest that paternal
genes play an important role in the development of pre-eclampsia. This has been
evidenced by the increased risk of pre-eclamptic women with pregnancies with men
who have previously been involved in pregnancies complicated with pre-eclampsia
(Valenzuela et al., 2012).
There is no cure for pre-eclampsia, with early delivery the only known ‘cure’
for pre-eclampsia, it is important that the maternal syndrome is managed to prolong
gestation whilst ensuring maternal safety. There have been various preventive
measures of treating pre-eclampsia undertaken in the past which includes
administration of calcium supplementation, anti-oxidants (vitamin C and E) and
aspirin (National Institute for Health and Clinical Excellence, 2010, Hofmeyr et al.,
2014). These approaches have initially showed promising results in small clinical trials
however findings were unable to be reproduced in larger clinical studies (Hawfield et
al., 2009). Oral antihypertensive drugs like methyldopa and labetalol are given in order
to reduce the blood pressure to < 160/110mm Hg (Myers et al., 2002). Continuous
fetal check is essential in the management of severe pre-eclampsia. Magnesium
sulphate is the drug used for the prevention of pre-eclampsia. The dose of 4g of
Magnesium sulphate is administered followed by a maintenance intravenous dose of
1g/hour usually for 24 hours post-delivery (Altman et al., 2002). However, there are
4
General Introduction
Chapter 1
adverse effects associated with Magnesium sulphate toxicity, which includes paralysis,
depression of respiratory drive and arrhythmias. Therefore, the only cure for preeclampsia is delivery.
1.4
PLACENTA AND PRE-ECLAMPSIA
The focus on the placental origin of pre-eclampsia led to the discovery that
defective placentation is a likely the cause of the maternal syndrome (Redman, 1991).
Placentation is defined as the process by which the placental trophoblast cells invade
the decidua and through to the uterine wall. During this process, extravillous
cytotrophoblast, invade and remodel the uterine spiral arteries of decidua and
myometrium causing hemodynamic changes essential to the establishment of
pregnancy and placentation (Wang et al., 2009). In many cases of pre-eclampsia, this
process is deficient. Cytotrophoblast invasion of spiral arteries is shallow and the
required tissue remodelling does not occur. Therefore the decidua and myometrium
segments of the spiral arteries remain Narrow and constricted and blood flow is limited
(Figure1.1). Although, the clinical symptoms of the disease usually don’t appear until
the third trimester of pregnancy, poor placentation during the first weeks of gestation is
an early event that underpins the subsequent aetiology of the syndrome (Merviel et al.,
2004).
5
General Introduction
Chapter 1
Figure 1.1 In normal pregnancy, cytotrophoblast invasion of maternal spiral arteries
results in adequate placental perfusion to sustain the growing fetus,
transforming the spiral arteries from small-calibre high resistance vessels
to high-calibre high volume vessels. In pre-eclampsia, there is limited or
shallow invasion of spiral arteries and they remain small caliber resistance
vessels (Wang et al., 2009).
6
General Introduction
1.5
Chapter 1
PLACENTAL ANATOMY
The placenta is a temporary organ, discoid in shape, which is about 15 to 25
centimetres in diameter and weighing 700-900 grams. Formed during implantation,
this organ plays a crucial role in supporting the nutritive, respiratory, and excretory
functions of the developing fetus and provides a mechanism for the chemical
communication between the fetal and maternal systems (Benirschke, 1998). The
placenta is comprised of two components -fetal and maternal. The fetal component is
derived from tissue that arises from the conceptus, namely the chorion. The ‘leafy’
region of the chorion known as the chorion frondosom is made of a chorionic plate and
finger-like projections of chorionic villi. The chorionic villi are the basic structures for
exchange between fetal and maternal blood in the placenta (Huppertz, 2008). The
maternal aspect of the placenta, the decidua basalis, is derived from the decidua
(Figure 1.2). The space between the fetal and maternal components of the placenta is
filled with maternal blood and is called the intervillous space. The branches of
chorionic villi project into the intervillous space and are bathed in maternal blood.
Some villi attach to the decidua basalis and these villi are referred to as the stem or
anchoring villi. The remaining villi that float freely in the maternal blood are termed
branch or free villi (Sood et al., 2006). The chorionic villi are covered by a continuous
fused cellular layer called the syncytiotrophoblast which forms the interface for blood
gas exchange, transfer of nutrients and removal of waste. Beneath the syncytium are
the underlying villous cytotrophoblast which are the progenitors for the syncytium.
The syncytium is in a constant state of renewal and turnover through fusion of
underlying cytotrophoblast with the covering syncytium and syncytial fragments being
shed from the chorionic villi into the maternal circulation (Burton et al., 2009).
7
General Introduction
Chapter 1
Figure 1.2 Structure of human placenta (Sood et al., 2006).
1.6
AETIOLOGY OF PRE-ECLAMPSIA
Deficient trophoblast invasion and failure of uterine spiral artery remodelling
are key features in the pathogenesis of pre-eclampsia (Steegers et al., 2010). Despite
the severity of the disease, no comprehensive theory or single factor has been
suggested to explain the pathophysiology of the disease. Substantial evidence suggest
that poor trophoblast invasion and defective remodelling of spiral arteries, which leads
to the fluctuating oxygen concentrations plays a key role in the pathophysiology of the
disease. The placental oxidative stress in the pathophysiology the disease is the major
focus of this thesis.
Oxidative stress is associated with a wide array of diseases including
cardiovascular diseases, cancer, renal diseases, ageing and pre-eclampsia (Giustarini et
al., 2009). Oxidative stress arises due to an imbalance between reactive oxygen species
(molecules with unpaired electrons) and the capacity of anti-oxidants to protect against
8
General Introduction
Chapter 1
oxidative damage. The term Reactive Oxygen Species (ROS) refers to a number of
free radical and non-radical derivatives of oxygen molecules. Excessive ROS causes
biological oxidation of lipids, proteins and DNA as well as compromising membrane
function leading to cellular damage and pathological processes. ROS arise from
various sources such as mitochondria, through electron leakage from the complexes of
the electron transport chain and cytochrome P450 enzymes (Murphy, 2009). The most
common reactive oxygen species are the superoxide radical, hydroxyl radical,
hydrogen peroxide, nitric oxide (Burton et al., 2011). Mitochondrial oxidative stress
occurs when an excess of electorns enter the electron transport chain (due to increase
metabolism and supply of NADH) or there is a limit in oxygen to accept the electrons
at Complex 4. When this occurs electron pressure in the electron transport chan
increases and electrons “leak” and partially reduce oxygen forming reactive oxygen
species. It has been estimated that this occurs around 3% in normal metabolism but can
increased dramtacially in pathological conditions.
Mitochondria generates ROS during oxidative metabolism through the oneelectron reduction of molecular oxygen (O2), forming superoxide anion (O2 −).
Complexes I, II, and III of the electron transport chain contain sites where electrons
can escape from the normal transport pathway and partially reduce oxygen, resulting in
the formation of superoxide (Figure 1.3). There have been reports which suggest the
link between abnormal mitochondrial function and ROS generation can cause localised
biological oxidation and further impair mitochondrial function. Additionally oxidative
damage affects replication and transcription of mitochondrial DNA which results in
decreased mitochondrial function that ultimately leads to increase in production of
ROS and further damage to mitochondrial DNA (Cui et al., 2012). There is a growing
9
General Introduction
Chapter 1
literature suggesting that mitochondrial ROS production is also involved in redox
signalling and dysfunctional mitochondria activate recovery pathways (Murphy et al.,
2013, Sabharwal et al., 2014). Placental mitochondrial dysfunction has been
implicated as an aetiological factor in the development of pre-eclampsia.
ROS generation contributes to mitochondrial membrane depolarisation, loss of
ATP production and opening of the mitochondrial transition pore, which leads to
apoptosis (Kalogeris et al., 2014). Substantial evidence suggests that ROS production
is increased in pre-eclamptic placenta and contributes to potential oxidative stress,
which causes cellular DNA damage, oxidation of important proteins and increased rate
of lipid peroxides (Hubel, 1999).
Figure 1.3 Production of ROS through mitochondrial electron transport chain.
Mitochondrial complexes I and II utilises electrons donated from
NADPH and FADH2 to reduce Coenzyme Q, where they are transferred
to cytochrome c. Electrons from cytochrome c is used by Complex IV to
reduce molecular oxygen to water. Complexes I, III and 1V produce a
proton electrochemical gradient which phosphorylates ADP synthase
(Hamanaka et al., 2010).
10
General Introduction
Chapter 1
Oxidative stress and an enhanced inflammatory state are features of the
maternal syndrome which are the characteristics of pre-eclampsia which occurs when
the production of ROS exceeds the levels of anti-oxidants. ROS generation is caused
due to hypoxia in placenta (Agarwal et al., 2012). Trophoblast cells are prone to
oxidative stress which causes increased cell death and placental turnover, the result of
which is the increased shedding of placental debris into the maternal circulation. This
debris is derived from paternal as well as maternal genes and is potentially antigenic
eliciting a hostile maternal immune response and subsequent endothelial dysfunction
which generates the symptoms of the disease (Sánchez-Aranguren et al., 2014).
Placental hypoxia and re-oxygenation, through oxidative stress may also stimulate
placental synthesis of cytokines e.g. Tumor necrosis factor-α, Interleukin (IL)-6 and
IL-10 (Hung et al., 2002) which further contribute to maternal endothelial cell
activation. The mechanism underlying the endothelial dysfunction is significantly high
concentrations of placental soluble fms-like tyrosine kinase 1(sFlt-1) in plasma of
women with pre-eclampsia. sFlt-1 binds to two angiogenic factors– vascular
endothelial growth factor and placental growth factor and it is significantly enhanced
in placental trophoblast due to hypoxia which is associated with oxidative stress
(Poston, 2006).
The human placenta is in a continuous state of growth and apoptosis and
syncytiotrophoblast microparticles are found in the blood of normal pregnant women.
In normal pregnancy, spiral arteries undergo substantial remodelling and trophoblast
cells penetrate into myometrium and there is an expanded blood flow to the placenta.
In many cases of pre-eclamptic pregnancies, the remodelling is shallow and the
maternal blood enters the intervillous space at higher pressure resulting in fluctuations
11
General Introduction
Chapter 1
in oxygen concentration. This can generate periods of hypoxia or ischemia followed by
reperfusion which is a well-recognised method for generating ROS (Powe et al.,
2011). The concentration and character of placental microparticles increases in women
with pre-eclampsia and they activate neutrophils and result in endothelium
dysfunction, which causes an inflammatory response (Sargent et al., 2003). In
conclusion, oxidative stress and endothelial activation, which causes immune
activation leads to further trophoblast turnover and contributes to the progression of
pre-eclampsia and leads to apoptosis (Figure 1.4). In complicated pregnancies like preeclampsia or intrauterine growth restriction (IUGR) the chances of placental apoptosis
is greater in the first trimester of pregnancy as there is reduction in number of
trophoblast cell within spiral arteries which causes poor trophoblast invasion in
myometrium This damage leads to hypoxia and high resistance blood flow as
determined by Doppler ultrasound waveforms in IUGR and severe pre-eclampsia
(Whitley et al., 2007).
Figure 1.4 Trophoblast cells inducing apoptosis by the secretion of TNF-α and
Indolamine 2, 3-dioxygenase (IDO) (Kaufmann et al., 2003).
12
General Introduction
1.7
Chapter 1
PLACENTAL OXIDATIVE STRESS AND APOPTOSIS
Apoptosis is the process of programmed cell death that is required for
maintaining homeostasis in normal cells and tissues. The morphological features of
apoptosis include chromatin and nuclear condensation, blebbing of the plasma
membrane and cell shrinkage. This causes cells to break into small membrane
fragments, called apoptotic bodies (Figure 1.5). The activation of intracellular cysteine
proteases such as caspases are involved in the induction of apoptosis (Reed, 2000).
Although, caspases play a central role in progression of apoptosis their initiation is
governed by other factors. The family of Bcl-2 proteins are known to play an
important role in the induction of apoptosis. Bcl-2 prevents the release of Cytochrome
c in mitochondria and also inhibits the activation of caspase 3 and 9 (Burlacu, 2003).
As previously mentioned, mitochondria are the major targets of production of ROS
and there is growing evidence to show that mitochondrial ROS participate in the
induction of apoptosis through the intrinsic pathway of activation. The intrinsic
pathway induces apoptosis through cytotoxic stimuli, which produce intracellular
signals that act on the targets of cells and begin mitochondrial events. Numerous
cytotoxic stimuli including ROS and other cytotoxic elements, and a lack of growth
factors initiate this pathway and directly damage cells by oxidizing DNA and
activating metalloproteinases. This results in the activation of pro-apoptotic proteins
which leads to changes in the inner mitochondrial permeability, loss of membrane
transmembrane potential, release of Cytochrome c to cytosol and opening of the
mitochondrial permeability transition pore which allows the activation of caspases
(Elmore, 2007, Kalogeris et al., 2014). The cell receptor mediated extrinsic pathway
involves activation of Tumor Necrosis Factor (TNF), Nerve Growth Factor (NGF) and
13
General Introduction
Chapter 1
Fas cell death receptors that result in signal transduction pathways causing apoptosis
(Simon et al., 2000). Since, ROS formation is significantly higher in mitochondria and
there have been considerable evidences in which apoptosis can be induced by ROS.
This triggers the apoptotic signal and cells sustain lipid peroxidation which leads to
oxidative stress (Matés et al., 2000). Previous studies suggest that apoptosis is induced
by free radicals and anti-oxidants have been shown or delay apoptosis. Apoptosis was
induced by hydrogen peroxide in mouse thymocytes and blastocysts that resulted in the
production of free radicals and ROS and apoptosis was inhibited by trolox, an inhibitor
of lipid peroxidation and glutathione and catalase (Forrest et al., 1994, Parchment,
1993). Thus, anti-oxidants play a key role in combating ROS and inhibiting apoptosis.
During pregnancy, apoptosis is important for normal placental development
and turnover however is also responsible for pathological conditions associated with
the placenta. As trophoblast cells undergo apoptosis, macrophages which are present in
maternal-fetal interface remove apoptotic cells by the process of phagocytosis
(Straszewski et al., 2005).
Figure 1.5 Morphological features of apoptosis (Microbiology Bytes: Virology, 2007).
14
General Introduction
1.8
Chapter 1
ANTI-OXIDANTS
In order to counteract intracellular damage by ROS, cells have developed an
intracellular anti-oxidant system. Oxidative stress occurs when the levels of ROS
exceeds the anti-oxidant capacities, such as in pre-eclampsia (Poston et al., 2011).
Anti-oxidants can be categorised into two major groups: exogenous which are also
called non-enzymatic or non-protein anti-oxidants, and endogenous anti-oxidants,
which are also known as enzymatic and are protein based. These low molecular weight
compounds scavenge super oxide and hydroxyl radical to produce hydrogen peroxide
(H2O2) and prevent lipid peroxidation (Stark, 2001).
The exogenous anti-oxidants cannot be produced by the body and are thus
obtained from diet, these include ascorbic acid, vitamin C, E, A and lycopenes. The
drawback feature of exogenous or non-protein based anti-oxidants is that the efficiency
of these anti-oxidants is very limited due to inability of these compounds to be
recycled (May et al., 1997).
Endogenous anti-oxidants on other hand are protein and enzymatic based
compounds synthesised by the body. The enzymatic based anti-oxidants include
superoxide dismutase (SOD), catalase, glutathione and thioredoxin systems. These
enzymes have very high turnover numbers and are able to negate the detrimental
effects of ROS in significant amounts. The deficiency of these micro-nutrients can lead
to poor pregnancy outcomes including fetal growth restriction and pre-eclampsia, and
additionally to cardio-vascular diseases and type-2 diabetes more broadly (Mistry et
al., 2011). Both the endogenous and exogenous systems play an important role in
protecting trophoblast cells from oxidative stress.
15
General Introduction
1.9
Chapter 1
MECHANISM OF ACTION OF ENDOGENOUS ANTI-OXIDANTS
The four major classes of endogenous anti-oxidants include Superoxide
Dismutase (SOD), catalase, glutathione and thioredoxin systems. Superoxide
Dismutase (SOD) forms the first line of defence against free radicals. SOD is present
in all aerobic cells and metabolizes two molecules of superoxide (O2-) to produce
H2O2. SOD exists in three different forms; copper, zinc isoform (CuZn-SOD, SOD1),
which is found in the cytoplasm, manganese superoxide dismutase (Mn- SOD2),
which is found only in mitochondria and extracellular SOD (SOD3), that contains
copper/zinc. All these three isoforms of SOD’S are found in placental tissues and
decreased levels of SOD have been associated with pre-eclampsia (Vanderlelie et al.,
2005). In pre-eclamptic pregnancy, a reduction in mRNA expression of placental SOD
has been observed, suggesting that changes in the SOD expression may underlie the
decreased activity seen in pre-eclamptic placental tissues (Wang et al., 2001).
Catalases are found in peroxisomes where they catalyses the conversion of
hydrogen peroxide to water and oxygen. Catalase is the powerful scavengers of free
radicals. Catalyses combine with phenols and alcohol to reduce H2O2 (Nordberg et al.,
2001). There have been studies which suggest that the levels of catalyse is significantly
lower in pre-eclamptic groups as compared to the healthy pregnant women (Wang et
al., 1996, Kolusari et al., 2008).
The Glutathione system consists of four components: Glutathione Peroxidase
(GPx), Glutathione (GSH), Glutathione Reductase (GR) and Glutathione-STransferase (GST). There are five isoforms of GPx, most of which contain
selenocysteine at the active site. The GPx1 and 4 are cystolic enzymes and are present
in most tissues, GPx2 is present in gastro-intestinal tract and GPx3 is expressed in
16
General Introduction
Chapter 1
kidneys. The GPx5 is expressed in epididymis and is selenium- independent (Nordberg
et al., 2001). GSH becomes oxidized when it reacts with hydrogen peroxides and lipid
peroxides by the action of GPx. During this process GPx becomes oxidized that
requires reduction by GSH, which oxidises to GSSG. GSSG is recycled to GSH by GR
in an NADPH-dependent reaction (Imai et al., 2003) (Figure 1.6).
Figure 1.6 Schematic summary of Glutathione-associated anti-oxidant systems
(Nordberg et al., 2001).
The Thioredoxin Peroxidase system consists of three anti-oxidant enzymes:
Thioredoxin Peroxidase (TPx), Thioredoxin (Thx) and Thioredoxin-Reductase (ThxRed). TPx catalyse the conversion of hydrogen peroxidase and alkyl hydroperoxides
into water and alcohols (Sen, 1998). There are five forms of TPx present in the
cytoplasm (TPx-1, 2, 4), mitochondria (TPx-3, 5) and secreted form (TPx-4)
(Nordberg et al., 2001).
Thioredoxin is a12-kDa protein with a highly conserved N-terminal active site
that contains two redox active cysteine residues in the sequence –Trp-Cys-Gly-ProCys-Lys-. Redox regulation by Thx occurs as a result of the reversible oxidation of the
active site dithiol to a disulphide resulting in oxidised thioredoxin (Thx-S2) that is
reduced via the action of Thx-Red in an NADPH dependant reaction (Figure 1.7).
17
General Introduction
Substrateox
Productred
Chapter 1
Thioredoxin
Peroxidase-SH
Thioredoxin-SH
Thioredoxin
Reductase-SH
Thioredoxin
Peroxidase S2
Thioredoxin S2
Thioredoxin
Reductase S2
NADPH
NADP+
Figure 1.7 Enzymatic reactions of Thioredoxin System (Nordberg et al., 2001).
1.10
ROLE OF ANTI-OXIDANTS IN PREGNANCY AND PRE-ECLAMPSIA
Oxidative stress is a key factor involved in the development of pre-eclampsia.
It has been suggested that taking anti-oxidants during pregnancy helps to scavenge
ROS, reducing oxidative stress and preventing or delaying the onset of pre-eclampsia
(Rumbold et al., 2008). Pathogenesis of pre-eclampsia is related to an imbalance
between oxidative stress and anti-oxidants (Burton et al., 2011). Substantial literature
suggests that adequate anti- defence may be protective against complicated pregnancy
like pre-eclampsia. It was demonstrated in one study that lower activity of SOD and
higher levels of catalase were associated in pre-eclamptic women as compared with
healthy pregnant controlled women (Dave et al., 2012). Reduced levels of SOD were
significantly associated with fetal death in pre-eclampsia (Rumiris et al., 2006).
Another study suggested that the activity levels of GPx and Thx-Red were deceased in
pre-eclamptic placentae (Vanderlelie et al., 2005). All these evidences confirm that
anti-oxidant enzymes are central to the pathophysiology of pre-eclampsia and thereby,
protecting trophoblast cells from oxidative stress. It has been suggested that certain
endogenous anti-oxidants such as GPx, Thx-Red and some selenoproteins are
dependent on micronutrient, selenium for normal functioning (Burk, 2002).
18
General Introduction
1.11
Chapter 1
SELENIUM (SE) AND SELENOPROTEINS
Three groups of selenium-containing proteins are known: specific selenium-
binding proteins, non-specifically incorporated selenium and specific selenocysteinecontaining selenoproteins (Behne et al., 2001). Selenium is an important constituent of
anti-oxidant enzymes, especially GPx, Thx-Red and some selenoproteins such as
Selenoprotein P and W. However, the functions of these selenoproteins are not
completely understood. In total humans have 25 selenoproteins genes encoded. GPx
and Thx-Red are the two endogenous anti-oxidant enzymes which contain
selenocysteine at their active site, so they require selenium in adequate amount that
functions as a redox centre (Rayman et al., 2000). It is essential for the activity and
expression of these proteins for the selenium to be present. Selenocysteine insertion in
these seleno-enzymes requires UGA codon, a process mediated by other
selenoproteins. In this respect, selenium availability is crucial for not only the
production of seleno-cysteine but also for the controlling the expression of these genes
(Hondal, 2005).
Selenium is an essential micro-nutrient necessary for normal growth and
reproduction in animals. Availability of selenium varies between areas and soil uptake.
The concentrations of selenium are typically measured in plasma, serum, whole blood,
urine and toenails (Mistry et al., 2011). The major sources of dietary selenium are
vegetables, flour, bread, Brazil nuts, meat and seafood. Selenium occurs in an organic
form, as Selenomethionine (SeMet) and inorganic form as Sodium Selenite (NaSe). It
enters the food chain through plants grown on selenium adequate soils, thus selenium
intake is directly proportional to the soil selenium status (Riaz et al., 2012).
19
General Introduction
Chapter 1
The Recommended dietary intake of selenium, according to the National
Institute of Health (USA) (2013) for men is 70µg/day and 60µg/day for women, with
additional 10µg/day when a woman is pregnant or lactating. A deficiency of selenium
leads to Keshan’s disease, when the intake is 20µg/day or less and excess levels of
selenium leads to toxicity of selenium called Selenosis. Selenium is an important
constituent of anti-oxidant enzymes, especially GPx and Thx-Red, which protects cells
against the deleterious effects of free radicals (Watson et al., 2012). Thus, the
relationship between oxidative stress, pre-eclampsia and selenium deficiency need
further research, so it is important to discuss them in detail.
Decreased levels of GPx and Thx-Red cause oxidative stress and there have
been reports which suggest that women who are pre-eclamptic and selenium deficient,
intake of selenium may be important to stop the progression or delay the onset of preeclampsia (Mistry et al., 2008). Several studies from Turkey, United States and
Australia suggest that placental tissues collected from the normal pregnancy and preeclampsia report a significant reduction in GPx and Thx-Red activity in pre-eclampsia
(Vanderlelie et al., 2011). If selenium deficiency is confirmed in women who are
suffering from complicated pregnancy like pre-eclampsia and this continues to be
linked with GPx, then more clinical trials are required on selenium supplementation in
pregnancy which could be beneficial in preventing pre-eclampsia.
1.12
PRE-ECLAMPSIA AND SELENIUM
Many studies have shown that selenium is correlated with the development of
pre-eclampsia. There has been significant decrease of selenium observed in plasma and
toenail samples of pre-eclamptic mothers (Rayman et al., 2003). This indicates that
20
General Introduction
Chapter 1
selenium levels in women before the diagnosis of pre-eclampsia may be important.
Several studies have shown a correlation between selenium status and adverse
pregnancy outcomes (Mariath et al., 2011). It was observed that women who have
suffered from miscarriage in their first trimester of pregnancy had significantly lower
levels serum selenium levels as compared to healthy pregnant women (Barrington et
al., 1996). Results found by Al-Kunani et al. in 2001 also found that significantly
lower hair selenium concentrations in women with repeatedly occurring miscarriages
when compared with non-pregnant women who had successful pregnancies. Decreased
selenium levels have also been associated with neural tube defects (Güvenc et al.,
1995, Cengiz et al., 2004). There have been evidences which suggest that poor
selenium status has detrimental maternal outcomes. Maternal plasma serum
concentrations are found to be reduced in the pre-eclamptic pregnancies (Atamer et al.,
2005, Mistry et al., 2008). Decreased selenium levels have been found in amniotic
fluid of pre-eclamptic women and babies born to pre-eclamptic mothers have shown
reduced umbical cord blood selenium levels (Dawson et al., 1999).
There are limited models of the disease, however removing selenium from the
diet of rats has been found to induce the symptoms as of pre-eclampsia (Vanderlelie et
al., 2004). The placenta from these pregnancies also showed reduced levels of GPx
and Thx-Red. However, in-vitro and in-vivo studies have showed that selenium
supplementation has direct capacity to improve the activity of endogenous anti-oxidant
enzymes protecting trophoblast cells from oxidative damage (Schnabel et al., 2008).
The importance of selenium to reproductive health may centre on the
incorporation of selenium in the form of selenocysteine in the active site of
endogenous anti-oxidant proteins such as GPx and Thx-Red to combat oxidative
21
General Introduction
Chapter 1
damage from ROS. However, other important selenoproteins may also have a role to
play in the aetiology of pre-eclampsia including selenoproteins P, W and S (Riaz et al.,
2012). Selenoprotein P plays a significant anti-oxidant function in protection of human
plasma from oxidation and nitration by peroxynitrite (Arteel et al., 1998). Recent
studies conducted on UK pregnant women suggested that Selenoprotein P
concentrations in plasma are also a useful marker of selenium status measured
throughout pregnancy (Rayman et al., 2014). There is a genetic association which
suggest that Selenoprotein S affects the risk of developing pre-eclampsia by increasing
inflammatory response (Moses et al., 2008). It has also been found that Selenoprotein
H, which is a DNA- binding protein, regulates the expression levels of genes which are
involved in glutathione synthesis and mitochondrial biogenesis (Mehta et al., 2013).
Studies done on Drosophila suggested that Selenoprotein H is crucial in
embryogenesis through its anti-oxidative activity (Morozova et al., 2003).
Selenoprotein H activates two transcription factors Peroxisome Proliferator-Activated
Receptor Coactivator-1alpha (PGC-1α) and Nuclear Respiratory Factor-1 (NRF-1)
through protein kinase A (PKA) and cAMP response element-binding (CREB)
pathways (Mehta et al., 2013). There have been reports which suggest that PGC-1α
and NRF-1 are important in maintaining fetal development (Pejznochova et al., 2010)
and the human placenta expresses PGC-1α. Interestingly, the expression of PGC-1α is
reduced in complicated pregnancies such as pre-eclampsia suggesting that PGC-1α
may be involved in the pathogenesis of complications of pregnancy such as preeclampsia (Delany et al., 2013).
22
General Introduction
1.13
Chapter 1
IN-VITRO MODELS OF TROPHOBLAST CELLS
Most of our knowledge about trophoblast cell behaviour has been derived from
in-vitro models which are required to study the preventive treatment before further
research is carried out. They are widely used in place of villous placental explants or
isolated primary cultures of trophoblast which are models of preference but more
difficult to establish due to ready access to human placental tissues. There are several
trophoblast cell lines that can be used for studying trophoblast cell biology most of
which have been isolated using various methods and modes of immortalization. One of
the most common features of the trophoblast cell line is the production of human
chorionic gonadotropin hormone (HCG). Choricarcinomic cell lines such BeWo, JEG3, Jar have been used for in-vitro studies. BeWo cells were established in 1968 after
being isolated from cerebral metastasis of choricarcinoma and then maintained in
hamsters for a number of years (Pattillo et al., 1968). These cell lines are more
frequently used as cell models as they show most of the features of villous trophoblast
which includes fusion and secretion of HCG. However, immortalization of cells in
many instances induces karyotypic and phenotypic abnormalities (King et al., 2000).
Therefore, the results from these studies, although valuable should be interpreted
cautiously. In order to overcome these limitations, Swan-71 cell lines have been
developed, which are non- choricarcinomic cell lines and isolated from primary
trophoblast cells from a 7-week normal placenta. The isolated cells were then
transfected with human telomerase reverse transcriptase (hTERT), the catalytic subunit
of telomerase (Straszewski et al., 2009) resulting in an immortalised trophoblast cell
line.
23
General Introduction
Chapter 1
The use of human placental villous explants to study the pathogenesis of preeclampsia has been extensive to validate the findings in other culture model. The
advantage of using explant models involving placental tissue can provide the
information by indicating how placenta behaves. Oxygen levels play a critical role on
the behaviour of both term and early placental explants. In the term placental culture,
ambient 3% O2 is associated with high levels of cytotrophoblast proliferation while
ambient 20% O2 can be associated with high levels of trophoblast apoptosis (Miller et
al., 2005). In our study, we have used first trimester and term tissues and both tissues
were cultured at 21% O2 (that is, ambient air). This is an experimental limitation
because placental tissue (particularly below 11 weeks) wouldn't be exposed to such
high oxygen tensions in- vivo. There is definitely damage to tissue during in-vitro
culture however; the placenta is still viable as tested through uptake of dyes that
require physiological processes (Chen et al., 2009). Studies can be done on explant
cultures derived from placentae which are associated with complicated pregnancies
such as pre-eclampsia, which can be useful for the patients who are at the risk of
disease.
1.14
SUMMARY AND RESEARCH PLAN
In conclusion, oxidative stress plays a significant role in the pathogenesis of
pre-eclampsia, which is a serious placental disorder affecting 5-7% of all pregnancies
leading to severe consequences for both mother and fetus. Trophoblast cells under
excessive oxidative stress results in the production of ROS and propagates apoptosis
and necrosis. Selenium supplementation has been shown to protect trophoblast cells
from ROS possibly because of its participation in the anti-oxidant defence system such
as GPx and Thx-Red. Thus, it was the objective of this thesis to demonstrate that
24
General Introduction
Chapter 1
selenium supplementation can up-regulate the key endogenous anti-oxidants enzymes
GPx and Thx-Red and whether selenium can protect trophoblast cells from
endogenous stressors in-vitro. As previously discussed that ROS stimulates apoptosis
and how selenium can delay or stop the induction of apoptosis was also a question of
investigation in this thesis. Since, ROS production is associated with mitochondria,
how selenium increases mitochondrial function of trophoblast cells and activates
mitochondrial biogenesis has been reported in Chapter 6 and 7 of this thesis. Hence, it
was the focus of this thesis how selenium can reduce the impact of pre-eclampsia that
can reduce burden on pre-mature babies and health care systems.
25
CHAPTER 2
GENERAL METHODS
General Methods
2.1
Chapter 2
CELL CULTURE
2.1.1 General cell culture
Three trophoblast-like cell lines were utilised in this series of experiments,
human choricarcinoma cell lines BeWo and JEG-3 obtained from the American Tissue
Culture Collection (ATCC, USA) and Swan-71 non-choricarcinomic cell lines
provided by Professor Gil Mor, Yale University, USA. Cells were cultured at 37°C
with 5% CO2 and 21% O2 in Dulbecco’s Modified Eagle Medium (DMEM) (GIBCO
Life Technologies, Australia) containing 10% fetal bovine serum and 500U/mL
penicillin–streptomycin (GIBCO Life Technologies, Australia). At the time of
completing these experiments we did not have access to a tri-gas incubator to limit the
amount of O2 present. Therefore all experiments were carried out with 5% CO2 in air
which equates to approximately 21% O2
Upon reaching 70-80% confluence, cells were split 1:3 into new 75cc flasks
(Cell Star, Greiner Bio-one). This was performed by removal of media and washing
the cells with 10 mL of Dulbecco’s Phosphate Buffered Saline (DPBS) (GIBCO Life
Technologies, Australia). The DPBS was aspirated and cells dislodged by the addition
of 5mL of 0.25% trypsin, EDTA (GIBCO Life Technologies, Australia) following 5
minute incubation at 37°C in 5% CO2. After incubation, 5mL of the DMEM was added
to the cell suspension to nullify the proteolytic effect of trypsin. Cells were then
dispersed into new 75 cc flasks, at a split ratio of 1:3, and incubated at 37°C in 5%
CO2 until 70-80% confluency was reached.
27
General Methods
Chapter 2
2.1.2 Cell Preparations
Cells were harvested from 75cc flasks after detachment as described above.
Cell number was determined using a Neubauer haemocytometer. This was performed
by mixing 50µL of cell suspension and 50µL of 0.4% Trypan blue in an Eppendorf
tube, subsequently 10µL was loaded into the cell chamber and viewed under a
microscope. Cells were seeded into 6 well, 12 well and 96 well sterile plates at a
density of 150000, 24000 and 10000 cells per well respectively and then subjected to
various treatments.
2.1.3 Cell Extraction
To extract cellular protein, the confluent monolayer from 75cc flasks was
collected after 24 hours of continuous culture in selenium supplemented media at
100nM Sodium Selenite (NaSe) and 500nM Selenomethionine (SeMet). The collected
cell pellet was resuspended in 300µL of cell lysis reagent (50mM Tris-HCl, 150mM
NaCl, 1% Triton and 1% Tween-20, pH7.4) and incubated on ice for 30 min. The
lysed cell suspension was centrifuged at 12000 rpm for 5min to pellet cell debris. The
protein-containing supernatant was removed and stored at -20°C until use.
2.2
BIOCHEMICAL METHODS
2.2.1 Protein Estimations
Principle
Quantification of the protein from tissue and cell extracts was performed using
a BCA Protein Assay kit (Pierce, Rockford, USA) following the manufacturers
28
General Methods
Chapter 2
protocol. This assay is based on bicinchoninic acid (BCA) for colorimetric detection
and quantitation of total protein in a sample at 562nm.
Procedure
A set of standards was prepared using Bovine Serum Albumin (BSA) over the
range 0-2000µg/mL. Using the microplate procedure, 200µL of working reagent was
added to each well with 20µL of each standard or 20µL of each sample was added in
triplicate. The plate was covered with foil and then incubated for 30 min at 37°C. The
absorbance was measured using Multiskan FC Plate reader (Thermo Scientific,
Australia) at a wavelength of 540nm. The unknown protein concentration was
determined using the standard curve from BSA standards by substituting the
absorbance into the linear line for the best standard curve as represented by y=mx+c.
Protein concentrations were expressed as mg of protein per mL of cell extract
(mg/mL).
2.2.2 Anti-oxidant Assays
2.2.2.1 Glutathione Peroxidase Assay
Principle
Glutathione Peroxidase (GPx) activity was quantified using an activity assay,
as first described by Flohe & Gunzler (Flohe et al., 1984). GPx catalyses the
reductions of tert-butyl hydrogen peroxide and in so doing become oxidised. Oxidised
GPx is regenerated by Glutathione (GSH) that in itself becomes oxidised to
Glutathione disulphide (GSSG). The GSSG is returned to GSH by the donation of two
29
General Methods
Chapter 2
electrons from Nicotinamide adenine dinucleotide phosphate (NADPH) via the
enzyme Glutathione Reductase (GR). The rate of oxidation of NADPH directly
correlates to GPx activity and can be measured spectrophotometrically at 340nm.
Procedure
An assay buffer solution of 250mM KPi and EDTA (pH 7.4), 1.0U/mL
Glutathione Reductase (Sigma Aldrich), 2mM Glutathione (Sigma Aldrich) and 0.3
mM NADPH (Sigma Aldrich) was prepared. 180µL of assay mix was transferred into
each well of a 96 well plate with an air blank. 30µL of cell extract for each sample was
added in triplicate with 5µL, 10µL, 15µL or 20µL of 20mU GPx used as positive
control. Wells containing only assay mix were used as negative control. To initiate the
reaction, 10µL of 10mM tert-butyl H2O2 was added to the wells containing cell extract
or positive control. The reduction of NADPH was measured spectrophotometrically at
340nm at 1 min time intervals for 10 min using a Multiskan FC plate reader (Thermo
Scientific, Australia). One enzyme unit of GPx is defined as the reduction of 1μmole
NADPH per minute. The absorbance values were plotted against time and change in
absorbance at 340nm. A standard curve was generated using GPx positive control and
the equation of the line y=mx+c was used to calculate the GPx concentration of each
unknown samples. The activity measured was expressed as units per milligram of
protein (U/mg of protein).
30
General Methods
Chapter 2
2.2.2.2 Thioredoxin-Reductase Assay
Principle
This assay measures the Thioredoxin-Reductase (Thx-Red) activity in tissue
and cell extracts in the presence of Thioredoxin (Thx). Thx-Red reduces Thx using
electrons from NADPH. The reduced Thx reduces protein disulphides present in
insulin to sulfhydryl (–SH) groups and the –SH groups further reduce 5, 5’- dithio-bis
(2-dinitrobenzoic acid) (DTNP) which produces a yellow product, 5-thio-2nitrobenzoic acid (TNB) that is measured at 412nm (Hill et al., 1997). Thx-Red
activity was also measured by the reduction of DTNP in absence and presence of
aurothiomalate. Aurothiomalate is a specific Thx-Red inhibitor, which inhibits the
activity of Thx-Red. The greater the activity of Thx-Red, there is increase in the
production of TNB which corresponds to a greater spectrophotometric absorbance.
Procedure
A commercial Thioredoxin-Reductase assay kit from Cayman Chemical
(Michigan, USA) was used to determine the activity in cells extracts. Each sample was
assayed in triplicates with 20μL of cell extract per determination. Briefly, 120μL assay
buffer, 20μL of sample with ± 20μL of Thx-Red inhibitor, sodium aurothiomalate
(provided in the kit) was added in each well of a 96-well plate. 20μL of rat liver ThxRed was used as a positive control. Wells containing only assay mix were used as
negative control or background wells. Wells were also loaded with only assay mix and
inhibitor. The reaction was initiated by adding 20μL of NADPH and 20μL of DTNP to
all the wells.
31
General Methods
Chapter 2
The microtiter plate was agitated for 10 seconds in the plate reader and the
absorbance was recorded every minute at 412nm using a Multiskan FC plate reader
(Thermo Scientific, Australia). The rate of change in absorbance at 412nm per minute
was determined by plotting the average values of time, which generated the slope rate.
The rate of change in absorbance at 412nm for background was calculated and this rate
was subtracted from all the wells including background and inhibitor wells. The
activity of Thx-Red was calculated by using DTNP coefficient of 6.75mM-1. The
results were expressed as units per milligram of protein (U/mg of protein).
2.3 ASSAYS FOR MEASURING CELL VIABILITY
2.3.1 3-[4, 5-dimethylthiazol-2-yl]-2, 5 diphenyl tetrazolium bromide (MTT)
Assay
Principle
The MTT assay is a colorimetric assay for assessing cell viability. This assay
utilises mitochondrial dehydrogenase enzyme from healthy cells to cleave the
tetrazolium rings of the faded MTT forming dark blue formazan crystals that are
impermeable to cell membrane which results in accumulation within viable cells
(Mosmann et al., 1983) (Figure 2.1).
32
General Methods
Chapter 2
Figure 2.1 MTT is converted into Formazan (Ebada et al., 2008) Enzyme
mitochondrial dehydrogenases metabolize MTT yellow dye to the
formation of MTT formazan blue crystals.
Procedure
Trophoblast cells were seeded into 96 well sterile plates at 10000 cells per well
in 150µL of DMEM and incubated at 37°C in 5% CO2 for 24 hours. Upon cell
attachment, wells were treated with varying concentrations of Antimycin (160µM,
240µM, 320µM, 400µM, and 480µM) and Rotenone (10μM, 20μM, 50μM and
100μM). The cells were left to incubate for 4 hours at 37°C in 5% CO2. Antimycin is a
chemical compound produced by Streptomyces kitazawensis. Antimycin is the
mitochondrial electron inhibitor which inhibits complex III of mitochondrial electron
transport chain by to binding to the Qi site of cytochrome c reductase and induces ROS
production (Chen et al., 2003). Rotenone blocks mitochondrial respiratory chain
complex I, thereby increasing the formation of ubisemiquinone, the primary electron
donor in mitochondrial superoxide generation (Li et al., 2003).
The cells were also treated with Sodium Selenite (NaSe) or organic
Selenomethionine (SeMet) at 100nM, 200nM, 400nM, 800nM, 1200nM, 1600nM,
33
General Methods
Chapter 2
2000nM for 24 hours at 37°C in 5% CO2. Assays were performed in triplicate and 25%
DMSO was used as a positive control. Post-incubation, the supernatant was removed
and cells were washed once with PBS. A mixture of 150µL of DMEM (without serum)
and 50µL of 1mg/mL MTT in PBS solution was added to each well. This was
followed by incubation for 2 hours at 37°C in 5% CO2. The supernatant was removed
and 125µL of DMSO was added followed by incubation for 45-60 min at room
temperature to dissolve the formazan. The absorbance was measured with a Multiskan
FC spectrophotometric microplate reader (Thermo Scientific, Australia) at a
wavelength of 560 nm. All the experiments in this thesis were performed using 100nM
NaSe and 500nM SeMet concentrations as these concentrations were able to increase
the expression and activity of key anti-oxidant proteins without inducing cytotoxicity
in trophoblast cell lines (described in Chapter 3 of this thesis).
2.3.2 Resazurin End Point Assay
Principle
The Resazurin end point assay measures the viability of mammalian cells and
bacteria. Living cells, which are metabolically active, are able to reduce the nonfluorescent dye resazurin to the strongly-fluorescent dye resorufin. The fluorescence
produced corresponds to the number of viable cells. Resazurin assay is an indicator of
oxidation-reduction which detects the generation of NADH/NADPH and also
determining the mitochondrial metabolic activity (Dukie et al., 2005).
34
General Methods
Chapter 2
Procedure
BeWo, JEG-3 and Swan-71 cells were seeded in 96 well sterile plates with a
cell number of 10000 cells per well in 150µL of DMEM and incubated at 37°C in5%
CO2 for 24 hours. After cell attachment, wells were supplemented with various
concentrations of Antimycin (160µM, 240µM, 320µM, 400µM, and 480µM) for 4
hours and incubated at 37°C in 5% CO2. The cells were also treated with Sodium
Selenite (NaSe) or organic Selenomethionine (SeMet) at 100nM, 200nM, 400nM,
800nM, 1200nM, 1600nM, 2000nM for 24 hours at 37°C in 5% CO2. Vacuum
aspiration was used to remove the media and cells were washed twice with DPBS.
Resazurin (200µL of 40nM) was added to each well followed by 2 hour incubation at
37°C. The amount of reduction of resazurin to resorufin was assessed by fluorescence
(excitation 530nm; emission 590nm) using a Fluoroskan Ascent microplate
fluorometer (Thermo Scientific, Australia).
2.4
ASSAYS FOR MEASURING OXIDATIVE STRESS
2.4.1 2’,7’-Dichlorofluorescein diacetate (DCFDA) Assay
Principle
2’,7’-Dichlorofluorescein diacetate is a fluorescent based dye, which measures
various reactive oxygen species, specifically hydroxyl and peroxyl reactive species
within cells. H2DCFDA is a non-fluorescent dye until it is cleaved by cellular esterases
and oxidation takes place in the cell (Eruslanov et al., 2010). The highly fluorescent
compound, DCF can be measured by fluorescence spectroscopy (Figure 2.2).
35
General Methods
Figure
2.2
Schematic
Chapter 2
representation
of
reactions
involved
in
2’,
7’
Dichlorofluorescein diacetate (DCFDA) Assay (Hensley et al., 2003).
Formation of fluorescent compound DCF by ROS.
Procedure
Swan-71 cells were seeded into black 96-well plates at a density of
10000cells/well. The cells were treated with 100nM Sodium Selenite (NaSe) or
organic Selenomethionine 500nM (SeMet). Cells were incubated for 24 hours at 37°C
in 5% CO2 to reach 60-70% confluency. Following cell attachment, media was
removed and cells were washed once with 100µL PBS. DCFDA dye was added at the
concentration of 50µM, prepared in DMSO. This solution was added to each well
using media without phenol red, followed by incubation for 45 minutes at 37°C in 5%
CO2. After treatment with the dye, media was removed and cells washed with 100µL
PBS. Cells were treated with Antimycin, at a concentration of 160µM, 240µM and
320µM or Rotenone concentrations of 10µM, 20µM, 50µM and 100µM and incubated
for 4 hours at 37°C in 5% CO2. Following incubation, fluorescence was measured at an
excitation 485nm and emission of 538nm using a Fluoroskan Ascent microplate
fluorometer (Thermo Scientific, Australia).
36
General Methods
2.5
Chapter 2
REAL TIME POLYMERASE CHAIN REACTION (PCR)
Principle
Real-time quantitative PCR is a reliable method of amplifying and
simultaneously detecting target gene expression. The products generated during each
cycle of the PCR process, are directly proportional to the amount of template present
prior to the start of the PCR process. A fluorescent signal is detected from the above
threshold which is considered a signal that can be used to define the threshold cycle
(Ct) for a sample.
Procedure
The RT-PCR was carried out using the following procedure; 25ng/μL of total
DNA was combined with 10μM of forward and reverse primers and assessed with 5x
Eva Green qPCR. SuperMix- Selenocysteine, Aspartic acid and Glycine (UDG) (Solis
BioDyne, Australia) on an Eco qPCR System (Illumina, Australia). The PCR protocol
consisted of a 95°C step for 15 minutes followed by 40 cycles, consisting of 15
seconds at 95°C, 20 seconds at 60°C and 20 seconds at 72°C followed by a melt curve
analysis. When RNA was converted from cDNA the protocol of PCR consisted of a
95°C step for 5 minutes followed by 40 cycles consisting of 30 seconds at 95°C, 45
seconds at 64°C and 30 seconds at 72°C followed by a melt curve analysis. The realtime PCR was performed three times using three biological replicates for each sample.
For each qPCR experiment, the samples were normalized to the reference β-actin gene
to give ΔCT for each gene. The specific primers used for RT-PCR are mentioned in
Table 6.1 (primers are designed by Truska et al., 2015, Venegas et al., 2011), Table
37
General Methods
Chapter 2
7.1 (primers are designed by Vanderlelie et al., 2008) and Table 7.2 (primers are
designed by Truska et al., 2015, Venegas et al., 2011).
2.6
cDNA SYNTHESIS
cDNA was synthesized from extracted RNA using QuantiTect Reverse
Transcription kit (Qiagen, Australia). Briefly, 1μg of purified RNA was combined with
7x genomic DNA Wipeout Buffer (Qiagen, Australia) with the final volume made up
to 14μL with RNase-free water. This mix was incubated for 2 minutes at 42°C on a
heating block and then immediately placed on ice. A reverse-transcription master mix
was prepared for each sample with 1μL of Quantiscript Reverse Transcriptase (Qiagen,
Australia), 4μL of 5x Quantiscript RT buffer (Qiagen, Australia) and RT Primer mix
(Qiagen, Australia). RNA template was added in each tube containing reversetranscription master mix and was incubated at 42°C for 15 minutes. Reaction mix was
further incubated for 3 minutes at 95°C to inactivate Quantiscript Reverse
Transcriptase and synthesized cDNA was stored at -20°C for further use.
2.7
WESTERN BLOTTING
BeWo, JEG-3 and Swan-71 (0.15x106) cells were cultured in 6-well plates
(Grenier Bio-One Cellstar) for 24 hours in 5% CO2 at 37°C incubator. The cells were
supplemented with Sodium Selenite (NaSe) at 100nM and 500nM Selenomethionine
(SeMet) for 24 hours. Trophoblast cells were treated with different concentrations of
Rotenone at 10μM, 20μM, 50μM and 100μM for 4 hours induce apoptosis. The
medium was aspirated and cells were washed once with phosphate buffer saline (PBS;
Gibco, Life Technologies, Australia). 40μL of lysis buffer (Cell Signalling
38
General Methods
Chapter 2
Technology, USA) was added to the cells with the addition of 1mM
phenylmethylsulfonyl fluoride (PMSF) and complete protease and phosphatase
inhibitor cocktail (Sigma Aldrich, Australia). Cells were incubated on ice for 10
minutes and were scraped away from the plastic surface of the plates with a cell
scraper (Grenier Bio-One Cellstar). Cell lysates were centrifuged at 12000x g for 10
minutes at 4ºC and the supernatant was collected to perform western blot analysis. The
protein concentration of each sample was determined by BCA calorimetric assay using
bovine serum albumin (BSA) assay kit (Pierce, Rockford, USA) as described in
section 2.2.1. 30μg of each protein extract was solubilised in gel-loading buffer (BioRad, Australia) and resolved by 4-15% SDS-PAGE on a Mini-PROTEAN® (Bio-Rad,
Australia). Multiple gels were run for different treatments. The resolved proteins were
transferred on to a Polyvinylidene fluoride (PVDF) membrane (Bio-Rad) by wettransfer method on a Criterion™ Blotter (Bio-Rad). After the protein transfer, PVDF
membranes were blocked with Tris-buffered saline (TBS; 50mM Tris-HCl, 150mM
NaCl, pH-7.4) with 5% BSA for 1 hour. The individual membranes were incubated at
4ºC with a primary antibody of interest dilution of 1:1000 (in TBS with 5% BSA and
0.1%Tween-20). This was followed by washing of the blots 3x for 10 min each using
Tris-buffered saline and Tween-20 TBST on a shaker. After this, the membranes were
incubated with horseradish peroxidase-conjugated secondary antibody at a dilution of
1:5000 for one hour at room temperature. Blots were again washed three times with
TBST. The blots were developed using the Clarity Enhanced chemiluminescence
(ECL) Western Substrate (Bio-Rad) and imaged. Densitometry analysis was performed
using Image J software (National Institute of Health, USA). Expression of proteins of
interest was first normalised to actin expression (protein content control), then the
39
General Methods
Chapter 2
relative expression of proteins of interest between untreated control samples and
treated samples was compared.
2.8
STATISTICAL ANALYSIS
The data presented in this thesis was processed using Graph Pad, PRISM
version 6 statistical package (GraphPad Software, San Diego California USA,
www.graphpad.com). All the data was represented as mean ± standard deviation.
Grubb’s test was used for identifying the outliers before analysis. Each data point was
determined in at least triplicate with all experiments performed a minimum of 3 times.
P values less than 0.05 were considered significant for all analysis.
2.8.1 One-way Analysis of Variance
One-way ANOVA test is performed to observe if there is any significant
difference between the treatments and non-treatment groups. Tukey’s post-hoc tests
are implied to determine which groups were significantly different from each other. A
One-Way Analysis of Variance is a way to test the equality of three or more means at
one time by using variances and then comparing the variance among these means to
the average variance within each group. The test statistic is thus the ratio of the
variance among means divided by the average variance within groups, or Fs (Chernick
et al., 2003). Post-hoc Tukey’s test is performed after an analysis of variance
(ANOVA) test. The purpose of Tukey's test is to determine which groups in the sample
differ and calculates the difference between the means and significance of all the
groups (Zar, 1999).
40
General Methods
Chapter 2
2.8.2 Two-tailed test
A two-tailed is a statistical test in which the area of distribution is two-sided.
This test is widely applicable with respect to the assumptions and normality. The twotailed measures if a sample is greater than or less than a certain range of values. This
test is also known as non-directional hypothesis (Zar, 1999). This test assumes the
differences between the pairs are normally distributed. Wilcoxon matched-pairs signed
rank test was performed to compare between the two samples.
41
CHAPTER 3
EXPRESSION OF GPx AND ThxRed IN RESPONSE TO SELENIUM
SUPPLEMENTATION
PUBLICATIONS ARISING FROM THIS CHAPTER
Khera A, Vanderlelie JJ, Holland O, Perkins AV. (2015) Expression of Glutathione
Peroxidase and Thioredoxin-Reductase in response to selenium supplementation.
Journal of Trace Elements in Medicine and Biology– Manuscript in preparation.
Expression of GPx and Thx-Red in response to selenium supplementation
3.1
Chapter 3
ABSTRACT
Endogenous anti-oxidant enzymes may contain trace elements copper,
manganese, zinc, and selenium that are essential to their function and activity. For
example, zinc is essential for functioning of more than 300 enzymes (Frassinetti et al.,
2006). In the present studies we investigated whether the addition of selenium, as
inorganic NaSe or organic SeMet, could increase the protein production and cellular
activity of the important redox active proteins Glutathione Peroxidase and
Thioredoxin-Reductase.
Placental trophoblast-like cell lines, BeWo, JEG-3 and Swan-71 trophoblast
cells were cultured in various concentrations of NaSe or SeMet for 24 hours. Cell
extracts and protein extracts were prepared for western blots and enzyme assays.
Selenotoxicity was also determined using MTT assays.
When trophoblast cells were supplemented with as little as 100nM NaSe and
500nM SeMet significantly enhanced the expression and activity of both GPx and
Thx-Red was noted. There was a clear dose dependent increase in enzyme activity up
to a maximum of 1μM. However, cytotoxicity data revealed a dose dependent increase
in the cellular toxicity of these compounds when tested at higher concentrations. These
observations are critical in order to set up the experimental models and to ensure
optimum cell culture conditions that allow for manipulation of both pro-oxidant and
anti-oxidant systems.
44
Expression of GPx and Thx-Red in response to selenium supplementation
3.2
Chapter 3
INTRODUCTION
The ability to modify the expression and activity of selected proteins in cell
culture is a central tool in studying cell biology. Mostly this entails transient gene over
regulation by inserting multiple copies of genes or using chemical enhancers of
expression to promote expression and boost endogenous production. An alternative
strategy is to knock down expression by silencing the expression of a particular gene
using chemicals or silencing RNA techniques. In some cases proteins require essential
trace elements to be active and this allows an alternative simple strategy to alter
production through trace element modulation as a method of control. This is the case
for many selenoproteins whose expression and activity is critically dependent on an
adequate supply of selenium (Sunde et al., 2011).
Selenium is an essential trace element, vital to many metabolic functions and
known for its anti-oxidant properties (Mistry et al., 2008, Tinggi, 2008, Hardy et al.,
2012). Despite its importance in human health, selenium is also known as a toxic
element that at high levels may contribute to reactive oxygen species formation and
significant impact cellular viability (Miller et al., 2011, Zou et al., 2007). In one study,
in HeLa cell lines, 5µM of selenite was found to be toxic and caused significant cell
death (Wallenberg et al., 2014). Therefore in this introductory work we examined the
toxicity of selenium in- vitro.
As described earlier selenium deficiency has been implicated in many
complications of human pregnancy (Pieczyńska et al., 2015) and the role of selenium
in placental biology is a central theme to this thesis. In this initial experimental work,
three different trophoblast-like cells lines have been used. BeWo and JEG-3 are
cancerous lines, whereas Swan-71 are a novel cell line developed in 2009, which is
45
Expression of GPx and Thx-Red in response to selenium supplementation
Chapter 3
non-cancerous and represents a truer model of human trophoblast (Straszewski et al.,
2009). In many other cell types selenium supplementation augments the expression
and activity of both GPx and Thx-Red but this has not been established in noncancerous cell lines such as Swan-71. Nor has a comprehensive study been completed
examining the dose response range of supplementation and how this influences
activity. The work in this Chapter investigates the expression and activity of the key
anti-oxidants, GPx and Thx-Red in response to selenium supplementation in -vitro.
46
Expression of GPx and Thx-Red in response to selenium supplementation
3.3
Chapter 3
AIMS
Experiments presented in this Chapter were required to set the conditions for
manipulating the expression of anti-oxidants and the generation of oxidative stress invitro.
The specific aims of this study were:
1 To determine the baseline levels of Glutathione Peroxidase (GPx) and
Thioredoxin- Reductase (Thx-Red) in trophoblast-like cells lines.
2 To examine the effect of selenium supplementation on the activity and
expression of Glutathione Peroxidase (GPx) and Thioredoxin- Reductase (ThxRed) in trophoblast-like cells lines.
3 To determine the toxicity of Sodium Selenite (NaSe), Selenomethionine
(SeMet), in trophoblast cells using 3-[4, 5-dimethylthiazol-2-yl]-2, 5 diphenyl
tetrazolium bromide (MTT) and Resazurin assays.
47
Expression of GPx and Thx-Red in response to selenium supplementation
3.4
Chapter 3
MATERIALS AND METHODS
3.4.1 Cell Culture plus Selenium supplementations
The choriocarcinoma cell lines BeWo and JEG-3 were obtained from American
Tissue Culture Collection (ATCC) and non-cancerous cell lines, Swan-71 were
provided by Professor Gil Mor, Yale University, USA. All the cell lines were grown in
Dulbecco’s Modified Eagle Medium (DMEM), (GIBCO Life Technologies, Australia)
in 5% CO2 and 21% O2 at 37°C containing 10% fetal bovine serum and 500U/ mL
penicillin–streptomycin (GIBCO Life Technologies, Australia). When confluent cells
were trypsinized and split in to new 75 cc flasks as described in section 2.1.1. BeWo,
JEG-3 and Swan-71 cells were also seeded in 96 well plates and adjusted to a cell
number of 10000 cells per well and 150μL DMEM was added to each well. Cells were
incubated for 24 h at 37°C. The cells were treated with both forms of selenium,
inorganic Sodium Selenite (NaSe) and organic Selenomethionine (SeMet) prepared in
media at various concentrations of 50nM, 100nM, 200nM, 400nM, 800nM, 1200nM,
1600nM and 2000nM. Cell viability assays were performed on these cells as described
in section 2.3.1 and 2.3.2.
3.4.2 Cell Extracts and Biochemical Analysis
Cellular proteins were extracted from BeWo, JEG-3 and Swan-71 cells,
supplemented with ± 100nM Sodium Selenite (NaSe) and 500nM Selenomethionine
(SeMet). Extracts for biochemical assays were collected from cells cultured in 75 cc
flasks after 24 h of culture in selenium supplemented media at 100nM NaSe and
48
Expression of GPx and Thx-Red in response to selenium supplementation
Chapter 3
500nM SeMet. Cells were lysed and protein extractions were performed on all cell
extracts as previously described in section 2.1.3.
3.4.3 Protein Estimations
Protein was quantified from the cell extracts using a BCA Protein Assay kit
(Pierce, Rockford, USA). Bovine Serum Albumin (BSA) standards were prepared and
using a microplate procedure, as described in detail in section 2.2.1. Twenty
microliters of each standard or 20μL of each sample was added in triplicate.
3.4.4 Glutathione Peroxidase Assay
The decrease of NADPH, which correlates Glutathione Peroxidase (GPx)
activity, was measured spectrophotometrically at 340nm in the cell extracts using a
Multiskan FC plate reader, as outlined in section in 2.2.2.1. The activity was measured
and expressed as units per milligram of protein (U/mg of protein). The inter assay and
intra assay coefficients of variation were 4.4% and 8.0% respectively.
3.4.5 Thioredoxin-Reductase Assay
The Thioredoxin-Reductase assay kit was purchased from Cayman Chemical
Company to determine the enzyme activity in cell extracts as per the manufacturer’s
protocol detailed in section 2.2.2.2. The inter assay and intra assay coefficients of
variation were 7.5% and 7.2% respectively.
49
Expression of GPx and Thx-Red in response to selenium supplementation
Chapter 3
3.4.6 Western Blotting
Swan-71 cells were seeded in a 6-well plate at a density of 0.15x106 and
incubated for 24 hours in 5% CO2 at 37°C. Cells were supplemented with Sodium
Selenite (NaSe) at 50nM, 100nM, 200nM, 400nM and 800nM concentrations for 24
hours. Western blotting was performed as described in section 2.7 on cell extracts after
measuring the protein concentration as outlined in section 2.2.1. Protein extracts
treated with 50nM, 100nM, 200nM, 400nM and 800nM NaSe were probed with
Glutathione Peroxidase 1 (Abcam, Australia), HRP-conjugated β-actin (Cell Signaling
Technology) and Thioredoxin- Reductase 1 antibody (Cell Signaling Technology) at
1:1000 dilutions overnight at 4°C shaker, followed by repeated washings with TBST.
The membranes were incubated with horseradish peroxidase-conjugated goat antirabbit secondary IgG antibody (Santa Cruz Biotechnology, USA) at a dilution of
1:5000 in TBST with 5% BSA for 1 hour at 37°C on a shaker. The blots were
processed as detailed in section 2.7 followed by densitometry analysis.
3.4.7 Statistical Analysis
All data was processed using Graph Pad Prism, version 6.0 with p<0.05
considered significant in all tests and all data is presented as mean +/- standard
deviation. One-way analysis of variance (ANOVA) with Tukey’s Post Hoc testing was
used to analyse significant differences between control and treatment groups as
described in detail in section 2.8.1. Each experiment was repeated a minimum of three
times with all experiments conducted in triplicate.
50
Expression of GPx and Thx-Red in response to selenium supplementation
3.5
Chapter 3
RESULTS
3.5.1 Expression of GPx protein in response to selenium supplementation
Protein extracts were prepared for Swan-71 cells after NaSe supplementation.
30μg of protein was loaded on 4-15% SDS-PAGE and western blotted onto PDVF
membranes. After probing with GPx antibody the membranes showed a dose
dependent increase in GPx protein expression up to a maximum with 800nM NaSe
(Figure 3.1 A). The fold increase at 800nM NaSe was an increase of 3.05 as compared
to the control (Figure 3.1 B). This increase was quantified using densitometry and
statistical analysis revealed significant increases in protein expression for cells
incubated with 100nM, 200nM, 400nM and 800nM concentrations of NaSe.
Figure 3.1 Expression of GPx protein in Swan-71cells when supplemented with
increasing amounts of NaSe (A). Each blot was repeated 4 times and a
representative blot is shown in panel A. Densitometry and statistical
analysis is shown in panel B. All data is presented as mean ± standard
deviation (S.D) (**p <0.01, ***p<0.001), CNTL= CONTROL.
51
Expression of GPx and Thx-Red in response to selenium supplementation
Chapter 3
3.5.2 Baseline activity of GPx in BeWo, JEG-3 and Swan-71 cell lines
The GPx activity was performed to determine the baseline enzyme activity.
The cells were harvested and lysed as described in section 2.1.1 and 2.1.3. In BeWo
cells, the baseline level of GPx activity was 1.356 ± 0.42 U/mg of protein (Figure 3.2).
There was a slight decrease in GPx basal level of Swan-71 cells (1.296 ± 0.157 U/mg
of protein) where as JEG-3 cells had the least baseline activity of 0.737 ± 0.034 U/mg
of protein. The assay was conducted using samples in triplicates and data presented is
from 5 different experiments.
Figure 3.2 Baseline activity of GPx in in BeWo, JEG-3 and Swan-71cell lines Values
are presented as mean ± standard deviation (S.D), n=5.
3.5.3 Dose dependent activity of GPx in Swan-71 in response to selenium
There was a dose dependent increase in activity of GPx when Swan-71 cell
lines were supplemented with varying concentrations of NaSe (50nM, 100nM, 200nM,
400nM, 800nM and 1200nM) and SeMet (100nM, 250nM, 500nM, 800nM, 1000nM
and 1500nM). When cells were supplemented with 1200nM NaSe GPx activity
reached at a maximal point of 3.784 ± 0.508 U/mg of protein (Figure 3.3 A). At
52
Expression of GPx and Thx-Red in response to selenium supplementation
Chapter 3
1000nM SeMet there was maximum GPx activity observed (3.034 ± 0.329 U/mg of
protein, Figure 3.3 B) and after that plateau was appeared.
Figure 3.3 Dose dependent activity of GPx in Swan-71, when supplemented with
NaSe (A) or SeMet (B) for 24 h treated with increasing concentrations of
selenium. Values are presented as mean ± standard deviation (S.D)
(*p<0.05, ** p<0.01***p<0.001, n=6).
3.5.4 Expression of Thx-Red protein in response to selenium supplementation
Assessment of selenium supplemental effects of Thx-Red expression was
carried out by running cell protein extracts on 4-15% SDS-PAGE and western blot
transfer was performed to PVDF membranes. After probing with Thx-Red antibody a
dose dependent expression of Thx-Red protein was observed (Figure 3.4 A).
Densitometry and statistical analysis showed significantly increased protein expression
at 400nM and 800nM NaSe with the fold increase of 5.23 and 7.19 as compared to the
cells which were not treated with selenium (Figure 3.4 B).
53
Expression of GPx and Thx-Red in response to selenium supplementation
Chapter 3
Figure 3.4 Expression of Thx-Red protein in Swan-71 cells when supplemented with
increasing amounts of NaSe. Each blot was repeated 4 times and a
representative blot is shown in panel A. Densitometry and statistical
analysis is shown in panel B as mean ± standard deviation (S.D) (**p
<0.01, ***p<0.001), CNTL= CONTROL.
3.5.5 Baseline Activity of Thx-Red in BeWo, JEG-3 and Swan-71 cell lines
Baseline activity of Thx-Red was quantified using the Thx-Red activity assay.
Cells were lysed and protein extracts were prepared. The protein concentrations were
quantified as described in section 2.2.1. The baseline rates of Thx-Red activity in
Swan-71 cells determined to be 0.012 ± 0.004 U/mg of protein. In BeWo cell lines the
baseline activity of Thx-Red activity was 0.016 ± 0.003 U/mg of protein. The highest
base level of Thx-Red activity was obtained in JEG-3 (as compared to BeWo and
54
Expression of GPx and Thx-Red in response to selenium supplementation
Chapter 3
Swan-71 cell lines), which reached up to significance (0.016 ± 0.003 U/mg of protein,
p<0.01, Figure 3.5).
Figure 3.5 Baseline activity of Thx-Red in BeWo, JEG-3 cell lines and Swan-71 cells.
Values are presented as mean ± standard deviation (S.D). Significant
differences were detected between control and maximal activity in 100nM
NaSe and 500nM SeMet (**p <0.01), n=5).
3.5.6 Dose dependent Activity of Thx-Red in Swan-71 cell lines supplemented
with increasing concentrations of selenium
The Thx-Red assay was performed to determine if NaSe and SeMet
supplementation could regulate the activity of Thx-Red in Swan-71 cells. Swan-71 cell
lines were supplemented with increasing concentrations of NaSe and SeMet. Swan-71
cell lines which were cultured with no selenium supplementation were considered as
control to determine the baseline levels of Thx-Red activity as 0.012 ± 0.004 U/mg of
protein. It was observed that Thx-Red activity increased with increasing concentrations
of both NaSe and SeMet. Maximum Thx-Red activity was observed when Swan-71
55
Expression of GPx and Thx-Red in response to selenium supplementation
Chapter 3
cells were supplemented with 800nM NaSe. When supplemented with 800nM NaSe,
the activity was measured as 0.056 ± 0.020 U/mg of protein (Figure 3.6 A). There was
a dose dependent increase in Thx-Red activity in Swan-71 cells, when they were
supplemented with increasing concentrations of SeMet. Maximum Thx-Red activity
was observed when cells were treated at 1500nM SeMet (0.0346 ± 0.072 U/mg of
protein) (Figure 3.6 B) The assay was repeated a minimum of three times with all the
samples assayed in triplicate.
Figure 3.6 Dose dependent activity of Thx-Red in Swan-71, when supplemented with
NaSe (A) or SeMet (B) for 24 h treated with increasing concentrations of
selenium. Values are presented as mean ± standard deviation (S.D)
(*p<0.05, ** p<0.01***p<0.001, n=6).
3.5.7 Effect of NaSe and SeMet toxicity on BeWo, JEG-3 and Swan-71 cells via
MTT
BeWo, JEG-3 and Swan-71 cells lines were cultured in media only or
supplemented with increasing concentrations of NaSe or SeMet at 100nM, 200nM,
56
Expression of GPx and Thx-Red in response to selenium supplementation
Chapter 3
400nM, 800nM, 1000nM, 1200nM and 1600nM for 24 hours in 5% CO2 at 37°C. The
negative control were cells cultured with no treatment of NaSe or SeMet and the
positive control used was 25% DMSO. The viability and cellular toxicity of both forms
of selenium in all trophoblast was assessed by MTT. There was a significant dose
dependent increase in the toxicity of both NaSe and SeMet, when measured by MTT.
The maximum NaSe toxicity in BeWo, JEG-3 and Swan-71 cell lines was observed
when cells were supplemented with 2000nM NaSe (0.090 ± 0.007, 0.114 ± 0.008,
0.140 ± 0.028) (p<0.001) (Figure 3.7), where less than 50% of cellular viability was
observed. Similar toxicity levels were observed in BeWo and JEG-3 cells at 2000nM
(p<0.001). The minimal toxicity in all trophoblast cell lines was observed in 100nM
and 200nM, where the viability was observed more than 50%. The concentrations
above 400nM of NaSe started showing the toxic levels, where cellular viability was
determined 0.364 ± 0.059 at 800nM NaSe. Similar observations were made when cells
were supplemented with SeMet (Figure 3.7). Supplementing cells above 400nM SeMet
started to have significant toxic levels. Cell treatments were assayed in quadruplicate
and data presented is from 6 different experiments.
57
Expression of GPx and Thx-Red in response to selenium supplementation
Chapter 3
Figure 3.7 Effect of NaSe (blue bars) and SeMet (violet bars) on cellular viability and
toxicity measured by MTT on BeWo, JEG-3 and Swan-71 cells. Values are
presented as mean ± standard deviation (SD). (***p<0.001, n=6).
3.5.8 Effect of NaSe and SeMet toxicity on BeWo, JEG-3 and Swan-71 cells via
Resazurin
Trophoblast-like cell lines were cultured in DMEM and supplemented with
NaSe or SeMet at 100nM, 200nM, 400nM, 800nM, 1000nM, 1200nM and 1600nM for
24 hours in 5% CO2 at 37°C. The negative control were cells cultured with no
treatment of NaSe or SeMet. The cellular toxicity of NaSe and SeMet in all
trophoblast-like cell lines was determined by resazurin assay. There was a significant
dose dependent increase observed in the cytotoxicity of all trophoblast-like cell lines.
58
Expression of GPx and Thx-Red in response to selenium supplementation
Chapter 3
The maximum cytotoxicity was observed when supplementation of 2000nM NaSe was
given in BeWo (2452 ± 281), JEG-3 (2967 ± 453) and Swan-71 (2014 ± 438.1) (Figure
3.8). Concentrations above 200nM NaSe started showing the toxic levels. Similar
observations were made when cells were supplemented with increasing doses of
SeMet.
Figure 3.8 Effect of NaSe (blue bars) and SeMet (violet bars) on cellular viability and
toxicity measured by Resazurin on BeWo, JEG-3 and Swan-71 cells.
Values are presented as mean ± standard deviation (SD). (***p<0.001,
n=6).
59
Expression of GPx and Thx-Red in response to selenium supplementation
3.5.9
Chapter 3
Up-regulation of GPx in BeWo, JEG-3 and Swan-71 cell lines in response
to selenium
The GPx activity assay was performed to determine the up-regulation in BeWo,
JEG-3 and Swan-71 cells. The cells were harvested and lysed as outlined in section
2.1.1 and 2.1.3. As shown in above results 3.5.7 and 3.5.8, trophoblast-like cell lines
started showing toxic levels above 200nM NaSe and 500nM SeMet, hence these
concentrations were chosen to determine if they can up-regulate the GPx activity in
trophoblast-like cell lines.
In BeWo cells the baseline level of GPx activity observed was 1.296 ± 0.151
U/mg of protein. After supplementation with NaSe (100nM) and SeMet (500nM), GPx
activity was increased to the level of 2.835 ± 0.142 U/mg and 2.737 ± 0.144 U/mg of
protein respectively (Figure 3.9 A). The JEG-3 cell line showed increase levels of
expression similar to that observed in BeWo cells (Figure 3.9 B). The baseline level of
GPx activity in JEG-3 cell lines was measured at 0.734 ± 0.0341 U/mg of protein. The
activity was significantly increased to 1.485 ± 0.0987 and 1.324 ± 0.5022 U/mg of
protein in JEG-3 cell lines supplemented with 100nM of NaSe and 500nM SeMet
respectively. There was significant increase in the GPx activity when Swan-71 cells
were supplemented with 100nM NaSe (1.727 ± 0.301 U/mg of protein) and 500nM
SeMet (2.336 ± 0.483 U/mg of protein, Figure 3.9 B). The assay was conducted using
samples in triplicates and data presented is from 5 different experiments.
60
Expression of GPx and Thx-Red in response to selenium supplementation
Chapter 3
Figure 3.9 Up-regulation of GPx activity in in BeWo (A), JEG-3 (B) and Swan-71cell
lines (C) when supplemented with 100nM NaSe and 500nM SeMet.
Values are presented as mean ± standard deviation (S.D). Significant
differences were detected between control and maximal activity in 100nM
NaSe and 500nM SeMet (*p<0.05, **p <0.01, ***p<0.00, n=5).
3.5.10 Up-regulation of Thx-Red in BeWo, JEG-3 and Swan-71 cell lines in
response to selenium
Thx-Red activity was quantified using the Thx-Red activity assay. Trophoblastlike cell lines were supplemented with 100nM NaSe and 500nM SeMet. The baseline
rate of Thx-Red activity in Swan-71 cells was 0.012 ± 0.004 U/mg of protein. When
supplemented with 100nM NaSe and 500nM SeMet (Figure 3.10 B), activity of ThxRed was significantly increased to 0.028 ± 0.006 and 0.22 ± 0.015 U/mg of protein
respectively (P<0.001). In BeWo cell lines the Thx-Red activity, when supplemented
with 100nM NaSe was found to be 0.072 ± 0.007 U/mg of protein. Thx-Red activity in
BeWo cells was also increased when treated with 500nM SeMet 0.069 ± 0.005 U/mg
protein (Figure 3.10 A). Activity of Thx-Red in JEG-3 cells increased significantly
61
Expression of GPx and Thx-Red in response to selenium supplementation
Chapter 3
when supplemented with 100nM NaSe and 500nM SeMet, the activity increased to
0.114 ± 0.007 and 0.106 ± 0.002 U/mg of protein respectively (Figure 3.10 A).
Figure 3.10 Up-regulation of Thx-Red activity in in BeWo, JEG-3 (A) and Swan71cell lines (B) when supplemented with 100nM NaSe and 500nM
SeMet. Values are presented as mean ± standard deviation (S.D).
Significant differences were detected between control and maximal
activity in 100nM NaSe and 500nM SeMet (*p<0.05, **p <0.01,
***p<0.00, n=5).
62
Expression of GPx and Thx-Red in response to selenium supplementation
3.6
Chapter 3
DISCUSSION
Placental oxidative stress is a key element in the pathogenesis of serious
complications of pregnancy such as pre-eclampsia (Sánchez- Aranguren et al., 2014).
There are various reports which demonstrate increased levels of ROS, oxidised lipids
and oxidised proteins in placental tissues from pregnancies complicated by preeclampsia (Lee et al., 2003, Noris et al., 2005, McMaster-Fay, 2008). Previous work
from this laboratory has shown that increased production of these oxidised
macromolecules occurs in the pre-eclamptic placenta and there is a decrease in
expression of anti-oxidant enzyme systems. In particular the activity of Glutathione
Peroxidase (GPx) and Thioredoxin-Reductase (Thx-Red) are significantly reduced in
placental tissues from pre-eclamptic mothers (Vanderlelie et al., 2005).
GPx and Thx-Red are selenoproteins, and critical components of two very
important anti-oxidant systems. The expression and activity of these proteins is
dependent upon the adequate supply of selenium. Previous studies in cells (Lymbury et
al., 2010) and animals (Vanderlelie et al., 2004) have shown that increased selenium
co-relates with increased expression and activity of the anti-oxidant proteins. This
raises the possibility of selenium supplementation as an effective means of increasing
anti-oxidant expression and limiting oxidative damage due to ROS generation. The
purpose of this study was to induce anti-oxidant expression in trophoblast cells and
establish cell models for subsequent study. It was important to establish effective
concentrations of selenium supplementation that could significantly increase antioxidant expression without compromising cell viability.
Two forms of selenium, organic selenomethionine (SeMet) and inorganic
Sodium Selenite (NaSe) were used to up-regulate the seleno-dependent anti-oxidant
63
Expression of GPx and Thx-Red in response to selenium supplementation
Chapter 3
enzymes in trophoblast-like cell lines BeWo, JEG-3 and Swan-71. Antibodies specific
for GPx and Thx-Red demonstrated that selenium supplementation increased the
cellular content of the proteins in all three cell types. Furthermore, enzyme assays for
GPx and Thx-Red were used to demonstrate the up-regulation of activity in trophoblast
cells when supplemented with NaSe and SeMet. GPx and Thx-Red assays compared
the baseline levels and up-regulation in BeWo, JEG-3 and Swan-71 cells.
Supplementing
cells
with
100nM
Sodium
Selenite
(NaSe)
and
500nM
Selenomethionine (SeMet), showed an increase in the expression of both GPx and
Thx-Red activity. Swan-71 cells required higher selenium supplementation to
maximise the GPx and Thx-Red activity as compared to BeWo and JEG-3 cell line.
These experiments allowed us to determine the minimal concentrations required to
induce a significant change in expression of these important anti-oxidant enzymes and
confirmed the selenium dependent up-regulation of GPx and Thx-Red in all three
BeWo, JEG-3 and Swan-71 trophoblastic cell lines.
Selenium was first described as a toxic element and its role in human health
was not established for many years. Indeed selenium is toxic and there have been
reports of seleno poisoning in humans and in livestock (Nuttall, 2006, MacFarquhar et
al., 2010) For this reason toxicity tests were performed on trophoblast cells to predict
the adverse effects of higher doses of selenium. Cell viability assays using 3-[4, 5dimethylthiazol-2-yl]-2, 5 diphenyl tetrazolium bromide (MTT) and Resazurin were
conducted to determine the cytotoxicity of selenium on BeWo, JEG-3 and Swan-71
cells. MTT assay is based on the conversion of MTT into formazan crystals, which
determines mitochondrial activity (Van Meerloo et al., 2011) where as Resazurin assay
64
Expression of GPx and Thx-Red in response to selenium supplementation
Chapter 3
is based on reduction of the non-fluorescent dye resazurin to the strongly fluorescent
dye resorufin (Dukie et al., 2005).
There was a dose dependent increase in the toxicity of both Sodium Selenite
(NaSe) and Selenomethionine (SeMet). MTT and Resazurin assays showed that
concentrations above 200nM NaSe and 500nM SeMet, induced cytotoxicity (greater
than 50%) in all three cell lines. Hence, 100nM NaSe and 500nM SeMet were chosen
as the best concentrations, which could induce the least cytotoxicity in trophoblast cell
lines whilst significantly increasing expression and activity of selenoproteins GPx and
Thx-Red. Consequently, all the experiments in this thesis have been performed with
100nM NaSe and 500nM SeMet.
Selenium is an essential micro-nutrient necessary for normal growth and
reproduction in animals. On a global scale, selenium availability in soil varies between
areas. Selenium concentrations are usually measured in plasma, serum, whole blood,
urine and toenails (Mistry et al., 2011). According to National Institute of Health
(NIH, USA) recommended dietary allowance for selenium intake is 70µg for men and
60µg for women on daily basis, with additional 5µg when a woman is pregnant.
Selenium is an important constituent of anti-oxidant enzymes, especially GPx and
Thx-Red, and selenium supplementation has been proven to enhance anti-oxidant
expression in trophoblast cells. Work done by Vanderlelie et al. in 2011, showed the
reduced plasma levels and its association with pre-eclampsia. Thus, selenium treatment
may be effective therapy in protecting placenta from oxidative stress during preeclampsia (Perkins, 2011), as it serves as a selenocysteine molecule in endogenous
anti-oxidants enzymes, GPx and Thx-Red.
65
Expression of GPx and Thx-Red in response to selenium supplementation
Chapter 3
Hence, in this chapter optimisations have been performed to develop methods
to maximise enzyme expression and activity using a simple trace element additive.
These investigations have been undertaken in this thesis to determine the experimental
and cell culture conditions which will allow further investigations on the beneficial
effects of selenium in trophoblast cells.
66
CHAPTER 4
SELENIUM AND
MITOCHONDRIAL OXIDATIVE
STRESS
PUBLICATIONS ARISING FROM THIS CHAPTER
Khera A, Vanderlelie JJ, Perkins AV. (2013) Selenium supplementation protects
trophoblast cells from mitochondrial oxidative stress. Placenta 34: 594-8.
Selenium and mitochondrial oxidative stress
4.1
Chapter 4
ABSTRACT
Placental oxidative stress has been associated with the pathogenesis of pre-
eclampsia, a disorder affecting approximately 7% of pregnancies. Placental trophoblast
cells are vulnerable to oxidative stress, which may lead to cell death and the symptom
cascade of pre-eclampsia. The aim of this investigation was to study mitochondrial
oxidative stress induced through the inhibition of the electron transport chain at
complex I and complex III by Rotenone and Antimycin respectively. The effect of
selenium supplementation in preventing oxidative stress in trophoblast cells was also
studied. Antimycin and Rotenone were found to induce the ROS when measured by
DCFDA assay and selenium supplementation was shown to reduce ROS production.
Cell viability as assessed by the MTT assay demonstrated a dose dependent decrease
in the cellular activity in BeWo, JEG-3 and Swan-71 when treated with increasing
concentrations of Antimycin or Rotenone. Through both MTT and Reszaurin assays it
was found that the viability of cells increased with prior incubation with Sodium
Selenite (NaSe) or Selenomethionine (SeMet) that these cells were protected from
oxidative stress and reduce the presence of ROS. This data suggests that selenoproteins
such as GPx and Thx-Red have an important role in protecting trophoblast
mitochondria from oxidative stress. This emphasises the importance of maintaining an
adequate selenium supply during pregnancy and especially in pregnancies complicated
by conditions such as pre-eclampsia.
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Selenium and mitochondrial oxidative stress
4.2
Chapter 4
INTRODUCTION
Oxidative stress is associated with a wide array of diseases including
cardiovascular disease, and renal disease, cancer, ageing and pre-eclampsia (Lobo et
al., 2010). Oxidative stress is defined as an imbalance between ROS and the capacity
of anti-oxidants to protect against oxidative damage (Betteridge, 2000). If an oxidative
insult persists, oxidised macromolecules such as lipids, proteins and nucleic acids are
released. Continuous or excessive oxidative stress may lead to autophagy, apoptosis
and necrosis with the associated release of cellular debris (Halliwell, 2007). The
placenta generates ROS which causes the oxidation of macromolecules, increases cell
turnover and contributes to the cellular debris released into the maternal circulation. In
complications of pregnancy such as pre-eclampsia, it has been shown that oxidative
stress increases the shedding of oxidised macromolecules as well as cellular debris
from placentae into maternal circulation (Burton et al., 2011, Rusterholz et al., 2011).
This in turn activates the maternal immune system and had been linked to endothelial
cell dysfunction.
Protection from reactive oxygen species and oxidative stress is provided by
anti-oxidants including the important endogenous anti-oxidant enzyme systems that
are present in all cells (Poljsak et al., 2013). These include enzymes involved in the
Glutathione and Thioredoxin reducing systems such as Glutathione Peroxidase (GPx)
and Thioredoxin-Reductase (Thx-Red). These two proteins are selenoenzymes and as
shown in Chapter 3 of this thesis that selenium supplementation is able to increase the
expression and activity of these proteins in many cell types, in animal studies and in
humans (Mistry et al., 2008, Vanderlelie et al., 2004, Tanguy et al., 2012).
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Selenium and mitochondrial oxidative stress
Chapter 4
Earlier work from our laboratory demonstrated that up-regulation of GPx and
Thx-Red using both inorganic Sodium Selenite and organic Selenomethionine was
able to protect trophoblast-like cells from exogenously added reactive oxygen species
(Watson et al., 2012). As an extension, in this chapter we investigated how the
generation of oxidative stress when applied endogenously by selectively blocking the
electron transport chain, could be prevented by selenium supplementation thereby
protecting trophoblast-like cells from mitochondrial oxidative stress.
71
Selenium and mitochondrial oxidative stress
4.3
Chapter 4
AIMS
The specific aims of this study were:
1
To demonstrate the production of Reactive Oxygen Species (ROS) in
trophoblast through Rotenone and Antimycin inhibition of the electron
transport chain by 2’, 7’-Dichlorofluorescein diacetate (DCFDA) assay.
2
To investigate the effect of selenium supplementation on ROS production.
3
To measure cell viability post Rotenone and Antimycin treatment with and
without sodium selenite supplementation by 3-[4, 5-dimethylthiazol-2-yl]-2, 5
diphenyl tetrazolium bromide (MTT) assay in trophoblast-like cells.
4
To measure cell viability post Rotenone and Antimycin treatment with and
without sodium selenite supplementation by the fluorescent Reszaurin.
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Selenium and mitochondrial oxidative stress
4.4
Chapter 4
MATERIALS AND METHODS
4.4.1 Cell Culture and Treatments
Trophoblast-like cells, BeWo, JEG-3 and Swan-71 were grown and maintained
at 37°C in 5% CO2 and 21% O2 in Dulbecco’s Modified Eagle Medium (GIBCO Life
Technologies, Australia) containing 10% fetal bovine serum and 500 U/mL penicillinstreptomycin (GIBCO Life Technologies, Australia) as described in section 2.1.1.
Trophoblast cells were seeded in 96 well sterile plates at a density of 10000 cells/well
and 150μL DMEM was added to each well. Cells were allowed to attach for 24 hours
at 37°C in 5% CO2. Selenium supplementation was administered for 24 hours in the
form of inorganic Sodium Selenite (NaSe) or organic Selenomethionine (SeMet)
prepared in DMEM and added at concentrations of 100nM or 500nM respectively.
Subsequently cells were incubated for 4 hours at 37°C in 5% CO2 and treated with
concentrations of Antimycin 10μM to 400μM or Rotenone 100nM to 100μM to induce
oxidative stress and. Cell viability and ROS assays were then preformed.
4.4.2
Quantification of ROS levels and selenium supplementation
The 2’,7’-Dichlorofluorescein diacetate (DCFDA) assay was used to measure
ROS production in trophoblast-like cells as outlined in section 2.4.1.
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Selenium and mitochondrial oxidative stress
Chapter 4
4.4.3 MTT and Resazurin end point assay for cell viability
MTT and Resazurin assays were performed as per the protocols outlined in
section 2.3.1 and 2.3.2 respectively to determine the Antimycin and Rotenone cellular
toxicity.
4.4.4 Statistical Analysis
All the data is presented as mean +/- standard deviation. Statistical analysis was
performed using GraphPad, PRISM version 6 for Windows, GraphPad Software, San
Diego California USA, www.graphpad.com. One-way analysis of variance (ANOVA)
with Tukey’s Post Hoc testing was used for analysis as detailed in 2.8.1. P values less
than 0.05 were considered significant. Each experiment was repeated at least three
times as denoted in the figure legends with all data points performed in quadruplicate.
74
Selenium and mitochondrial oxidative stress
4.5
Chapter 4
RESULTS
4.5.1 ROS Production in Trophoblast cells post Rotenone and Antimycin
treatment
Swan-71 cells lines were treated as described above and DCFDA assays were
conducted to determine the ROS levels post Rotenone and Antimycin treatment.
Trophoblast cells treated with both mitochondrial inhibitors demonstrated a significant
(P<0.001) and dose dependant increase in reactive oxygen species when measured
with DCFDA. There was a dose dependent increase in ROS production with maximum
observed at 100μM Rotenone (4960 ± 488, Figure 4.1 A) and 320μM Antimycin (7033
± 949.6, Figure 4.1 B).
Figure 4.1 Effect of Antimycin (A) and Rotenone (B) on ROS production as compared
to control in Swan-71 cells. Values are presented as mean ± standard
deviation (S.D) (***p<0.001, n=6).
75
Selenium and mitochondrial oxidative stress
Chapter 4
4.5.2 Selenium supplementation reduces ROS production induced by Rotenone
and Antimycin
Swan-71 cells were cultured in the presence of NaSe at 100nM and SeMet at
500nM was for 24 hours at 37°C in 5% CO2. Subsequently, cells were stressed with
various doses of Rotenone at 10µM, 20µM ,50µM and 100µM and Antimycin at
160µM, 240µM and 320µM for 4 hours. ROS levels were quantified by DCFDA assay
(Figure 4.2 A-D). Cell treatments were assayed in quadruplicate and data presented is
from 6 different experiments.
Figure 4.2 Effect of NaSe supplementation and SeMet treatment on ROS production
with Antimycin (A, B) and Rotenone (C, D) treatment for 4 hours. Values
are presented as mean ± standard deviation (S.D) (***p<0.001, n=6).
76
Selenium and mitochondrial oxidative stress
Chapter 4
Swan-71 cells treated with Rotenone and 100nM NaSe showed a significant
decrease in ROS levels at 100μM (2350 ± 53.20), 50μM (2594 ± 75.64), 20μM (1886
± 517.6) and 10μM (1072 ± 158.4) (Figure 4.2 A). There was also a decline in ROS
levels when supplementated with SeMet (Figure 4.2 B). Cells treated with no selenium
and no added stressor were considered as a contol and displayed a minimal amount of
ROS production. Supplementation with 100nM NaSe resulted in significant reduction
in ROS production at 320μM Antimycin (4435 ± 885.9) as determined by DCF
flurorescence. Reduced ROS production was also seen at 160μM Antimycin (3487 ±
1524) and 240μM Antimycin (4461 ± 726.4) (Figure 4.2 C). Identical observations of
a reduction in ROS levels was made when Swan-71 cells were supplemeted with
500nM SeMet (Figure 4.2 D).
4.5.3 Cellular viability measured by MTT in Swan-71 cells when treated with
Rotenone and effect of NaSe supplementation
Swan-71 cells were cultured in DMEM only or treated with 100nM NaSe for
24 hours and later exposed to Rotenone treatment at 10μM, 20μM, 50μM and 100μM
for 4 hours and the cytotoxicity was determined by MTT assay. Toxicity levels started
to increase after a concentration of 10μM. The maximum toxicity of Rotenone was
observed when cells were treated at 100μM (0.04 ± 0.008) (Figure 4.3).
Supplementation with 100nM NaSe showed significant protection (P<0.001) of Swan71 cells from cell death due to ROS generated by Rotenone. At 100μM, cells showed a
MTT reading of A560 0.09 ± 0.02 as compared to the non-selenium supplementation
(A560 0.04 ± 0.008, P<0.001 Figure 4.3). Similar observations were made at 20μM and
50μM of Rotenone treated cells after supplementing with selenium (Figure 4.3). These
77
Selenium and mitochondrial oxidative stress
Chapter 4
experiments were repeated in other trophoblast cell types and with similar
observations.
Figure 4.3 Effect of Rotenone cellular viability on Swan-71 cells measured by MTT
assay in addition with 100nM NaSe supplementation. Values are presented
as mean ± standard deviation (S.D) (***p<0.001, n=6).
4.5.4 Cellular viability measured by MTT in Swan-71 cells when treated with
Antimycin and effect of NaSe supplementation
The viability and cellular toxicity of Antimycin in combination with 100nM
NaSe treatment was assessed in Swan-71 cells using the MTT assay to observe the
impact of selenium. Cells cultured with Antimycin with no treatment with NaSe were
the negative control and 25% DMSO was used as a positive control. It was observed
that there was a dose dependent increase in the cellular toxicity of Antimycin
(P<0.001, Figure 4.4). Prior supplementation with 100nM NaSe for 24 hours was
introduced in combination with all Antimycin treatments with the aim of showing the
inhibitory effect of selenium on Antimycin toxicity.
78
Selenium and mitochondrial oxidative stress
Chapter 4
Figure 4.4 Effect of Antimycin on cellular viability of Swan-71 cells measured by
MTT assay in addition with 100nM NaSe supplementation. Values are
presented as mean ± standard deviation (S.D) (***p<0.001, n=6).
It was observed that selenium supplementation reduced the production of ROS
generated by Antimycin. Cells treated with Antimycin at 100μM that also received
NaSe supplementation showed an MTT reading of A560 0.10 ± 0.01 compared to those
that didn’t receive NaSe treatment (A560 0.04 ± 0.01 Figure 4.4). Similarly, the cells
treated with 50μM Antimycin had an absorbance of A560 0.06 ± 0.01 which increased
to A560 0.10 ± 0.011 when they received NaSe treatment (P<0.001). 10μM and 20μM
of Antimycin concentration also showed the similar protection levels with selenium
from ROS. Again these experimental conditions were applied to other trophoblast cells
and similar observations were made.
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Selenium and mitochondrial oxidative stress
Chapter 4
4.5.5 Cellular activity in BeWo, JEG-3, Swan-71 when oxidatively stressed with
Rotenone as assessed with Reszaurin
BeWo, JEG-3 and Swan-71 cell lines were grown in DMEM in 5% CO2 at
37°C for 24 hours and supplemented with 100nM, 200nM, 400nM, 600nM, 800nM of
Rotenone for 4 hours. Cellular activity was assayed using the resazurin end point
assay. To determine 100% cellular activity, no rotenone stress was added into the
control cell lines. There was a dose dependent decrease in the cellular activity in all
three cell types and with a minimal effect at 100nM (71.72 ± 26.03%, Figure 4.5 A)
and a maximal effect with 800nM Rotenone. Similar patterns were observed in JEG-3
and Swan-71 cells (Figure 4.5 B, C). At higher concentrations of 400nM and 800nM
there was a decrease in the cellular activity (24.60 ± 11.84%, 7.137 ± 6.428%)
respectively in JEG-3 cells (Figure 4.5 B). Treatment with 800nM of Rotenone appears
to have caused cell death and the cellular activity was measured as 18.44 ± 15.18% in
BeWo cell lines, similar effect was seen in JEG-3 and Swan-71 cell lines.
Figure 4.5 Effect of Rotenone on cellular activity of BeWo (A), JEG-3 (B) and Swan71 (C) cell lines respectively. Values are presented as mean ± standard
80
Selenium and mitochondrial oxidative stress
Chapter 4
deviation (SD). (*p<0.05, ***p<0.001, n=6.). Samples used in the assay
were conducted in quadruplicate.
4.5.6 Cellular activity of Antimycin in BeWo, JEG-3 and Swan-71 cells assessed
via Resazurin
BeWo, JEG-3 and Swan-71 cell lines were cultured in media and stressed with
increasing doses of Antimycin for 4 hours (40µM, 80µM, 120µM, 160µM, 240µM and
320µM). Control cell lines were cultured with no added stressors to determine 100%
cellular activity.
The resazurin end point assays was conducted to determine the effect of
oxidative stress on cellular activity in BeWo, JEG-3 and Swan-71 cell lines. Antimycin
concentrations of 80µM to 320µM applied for 4 hours, resulted in a dose dependent
significant decrease in cellular activity in all three cell types (P<0.0001) (Figure 4.6 A,
B, C). The higher concentrations of Antimycin at 320µM resulted in cell death and
maximum reduction in cellular activity. The reduction in cellular activity (less than
50%) was observed at 320µM (10.65 ± 7.739%, 11.30 ± 8.613%, and 9.155 ± 11.15%)
in BeWo, JEG-3 and Swan-71 cell lines respectively.
81
Selenium and mitochondrial oxidative stress
Chapter 4
Figure 4.6 Effect of Antimycin on cellular activity of BeWo (A), JEG-3 (B) and
Swan-71(C) cell lines respectively when they are oxidatively stressed.
Values are presented as mean ± standard deviation (SD). (*p<0.05, p**
<0.01, ***p<0.001, n=6) .Samples used in the assay were conducted in
quadruplicate.
4.5.7 Sodium Selenite (NaSe) protection from oxidative stress induced by
Rotenone
Trophoblast cells were grown in DMEM only or treated with 100nM NaSe for
24 hours and subsequently treated with increasing concentrations of Rotenone.
Supplementation with 100nM NaSe showed significant capacity to protect trophoblast
cells from oxidative stress generated by Rotenone. At a concentration of 200nM
Rotenone, cellular activity in selenium supplemented Swan-71 cells was significantly
increased as compared to non-selenium supplemented cells (105 ± 9.798% vs 71.94 ±
29.07% respectively) (P<0.001) (Figure 4.7 C). Supplementation of 100nM NaSe
82
Selenium and mitochondrial oxidative stress
Chapter 4
appeared to be effective as JEG-3 cells when treated with 200nM Rotenone the cell
activity of selenium supplemented cells showed 92.53 ± 19.66%, as compared to nonsupplemented selenium cells 76.50 ± 27.63% (Figure 4.7 B). Similar observations
were made in BeWo cells (Figure 4.7 A).
Figure 4.7 Effect of Rotenone on percentage cellular activity in BeWo (A), JEG-3 (B)
and Swan-71 cells (C), treated with NaSe for 24 hours and subsequently
treated with increasing doses of Rotenone concentration. Values are
83
Selenium and mitochondrial oxidative stress
Chapter 4
presented as mean ± standard deviation (* p<0.05, ** p<0.01, ***p<0.001,
n=6). Samples used in the assay were conducted in quadruplicate.
4.5.8 Selenomethionine (SeMet) protection from oxidative stress induced by
Rotenone
BeWo, JEG-3 and Swan-71 cells were grown in DMEM only or supplemented
with 500nM SeMet for 24 hours. Following attachment, cells were stressed with
increasing doses of Rotenone (100nM, 200nM, 400nM and 800nM) for 4 hours. There
was no difference between supplemented and un-supplemented cells that received no
stress treatment and these were set as 100% cell activity. Treatment with SeMet in
BeWo cells was found to protect against oxidative stress generated at 200nM and
400nM and showed activities of 101.3 ± 15.96%; P<0.05, 66.92% ± 26.20 %; P<0.001
respectively (Figure 4.8 A). JEG-3 also demonstrated a dose dependent decline in
cellular activity (Figure 4.8 B). Cell activity at 200nM and 400nM was 76.50 ±
27.63% and 24.60 ± 11.84% respectively, however when supplemented with organic
SeMet at these concentrations, cellular activities were significantly increased to 99.74
± 15.40% and 61.75 ± 13.26 respectively. Similar observations in cellular activities
were made in Swan-71 cells (Figure 4.8 C).
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Selenium and mitochondrial oxidative stress
Chapter 4
Figure 4.8 Effect of Rotenone on percentage cellular activity in BeWo (A) JEG-3 (B)
and Swan-71 cells (C), treated with SeMet for 24 h and later treated with
increasing doses of Rotenone concentration. Values are presented as mean
± standard deviation (* p<0.05, ** p<0.01, ***p<0.001, n=6). Samples
used in the assay were conducted in quadruplicate.
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Selenium and mitochondrial oxidative stress
Chapter 4
4.5.9 Sodium Selenite (NaSe) protection from oxidative stress induced by
Antimycin
BeWo, JEG-3 and Swan-71 cells cultured in media only or supplemented with
100nM NaSe and subsequently stressed with increasing doses of Antimycin (40µM,
80µM, 160µM, 240µM and 320µM) for 4 hours. There was no significant difference
between supplemented and un-supplemented cells that were not exposed to stress
treatment and these were set as 100% cellular activity. At concentrations of Antimycin
160µM, with NaSe treatment, significantly increased cellular activity from 42.18 ±
18.62% to 62.48 ± 18.62% (Figure 4.9 A). A similar pattern was observed in JEG-3
cells (Figure 4.9 B). In Swan-71 cells, significant differences were observed between
cells treated with Antimycin at 80µM, 120µM, 160µM and 240µM and those
supplemented with 100nM NaSe at the same concentration (Figure 4.9 B). At 80µM
Antimycin concentration, supplemented with 100nM NaSe the cellular activity
observed was 86.26 ± 16.45% as compared to non-treated NaSe cells with only
Antimycin 52.60 ± 23.57%; P<0.001). Similarly, at 120µM, 160µM and 240µM
Antimycin concentration supplemented with NaSe showed cellular activity 75.06 ±
9.47%, 78.16 ± 21.72% and 53.74 ± 22.88% as compared to non-NaSe supplemented
with the same concentrations which had 47.03 ± 12.09%; P<0.01, 26.74 ± 21.86%;
P<0.001 and 11.51 ± 9.96%; P<0.001 respectively (Figure 4.9 C).
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Selenium and mitochondrial oxidative stress
Chapter 4
Figure 4.9 Effect of Antimycin on percentage cellular activity in BeWo (A), JEG-3
(B) and Swan-71 cells (C), treated with NaSe for 24 hours and
subsequently treated with increasing doses of Antimycin concentration.
Values are presented as mean ± standard deviation (* p<0.05, ** p<0.01,
***p<0.001, n=6). Samples used in the assay were conducted in
quadruplicate.
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Selenium and mitochondrial oxidative stress
Chapter 4
4.5.10 Selenomethionine (SeMet) protection from oxidative stress induced by
Antimycin
Trophoblast cell lines were cultured in DMEM or supplemented with organic
form of selenium 500nM SeMet for 24 hours and later stressed with increasing
concentrations of Antimycin for 4 hours. There was no difference between
supplemented and un-supplemented cells that received no stress treatment and these
were set as 100% cellular activity. BeWo, JEG-3 and Swan-71 cells treated with
80µM, 120µM, 160µM, 240µM and 320µM Antimycin. Swan-71 cells showed a
significant dose dependent decrease in the cellular activity, when treated with 80µM,
120µM, 160µM, 240µM and 320µM Antimycin 52.60 ± 23.57%, 47.03 ± 12.09%, and
26.74 ± 21.86 %, 11.51 ± 9.99% respectively. Cells treated with the same
concentrations of Antimycin and then supplemented with 500nM SeMet showed
cellular activities 73.47 ± 34.15%, 84 ± 22.06%, 66.40 ± 27.35% and 65.97 ± 12.71%
(Figure 4.10 C). Significant differences were detected between supplemented and nonsupplemented cells with 500nM SeMet in BeWo (Figure 4.10 A) and JEG-3 cells
(Figure 4.10 B).
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Selenium and mitochondrial oxidative stress
Chapter 4
Figure 4.10 Effect of Antimycin on percentage cellular activity in BeWo (A) JEG-3
(B) and Swan-71 cells (C), treated with SeMet for 24 h and later treated
with increasing doses of Antimycin concentration. Values are presented
as mean ± standard deviation (* p<0.05, ** p<0.01, ***p<0.001, n=6).
Samples used in the assay were conducted in quadruplicate.
89
Selenium and mitochondrial oxidative stress
4.6
Chapter 4
DISCUSSION
Previous
work
in
our
laboratory
has
demonstrated
that
selenium
supplementation can protect trophoblast cells from exogenously applied oxidative
stress (Watson et al., 2012). Various forms of peroxides were used to stress cells in
this study and it was demonstrated that the up-regulation of GPx and Thx-Red through
selenium supplementation could protect trophoblast cells (Watson et al., 2012).
However these experimental observations may be criticised as the concentrations of
peroxides used were higher than would be present in- vivo and some of the peroxides
used such as cumene hydroperoxide would not occur naturally in cells. In this study,
endogenous oxidative stress was generated by blocking the electron transport chain
through Antimycin and Rotenone which cause electron leakage from the mitochondrial
electron transport chain and thereby result in the generation of superoxide (O2*-) in the
mitochondrial matrix that leads to generation of Reactive Oxygen Species (ROS) and
in effect mimic the effects of oxidative stress in-vivo. Rotenone and Antimycin block
Complex I and III of the electron transport chain respectively, resulting in the
generation of superoxide (O2*-) in the mitochondrial matrix that leads to generation of
ROS (Jastroch et al., 2010, Stowe et al., 2009). Due to high concentration and catalytic
activity of Mn-SOD, the O2*- is converted to H2O2 which is potentially very damaging
to cell viability (Cabiscol et al., 2000). This represents a more authentic method of
generating peroxide base oxidative stress, which permits the examination of the effect
of selenium supplementation in a model of endogenously produced oxidative stress.
Cell viability studies with Antimycin and Rotenone were conducted in BeWo,
JEG-3 and Swan-71 cell lines through the MTT and Resazurin assays. A dose
dependent increase in the toxicity of both Antimycin and Rotenone was observed
90
Selenium and mitochondrial oxidative stress
Chapter 4
presumably due to the increased generation of ROS as shown in Figure 4.1. Rotenone
and Antimycin both induced ROS production in the mitochondrial respiratory chain at
complex I and complex III respectively and ROS production was measured through the
DCFDA assay. ROS production was significantly higher at 320µM Antimycin and
100µM Rotenone.
Selenium treatment significantly improved the viability of trophoblast-like cells
when a mitochondrial specific oxidative insult was applied as selenium
supplementation has been shown to increase the activity of selenium based antioxidant enzymes in order to reduce endogenous ROS (Chapter 3). Trophoblast cells
supplemented with both inorganic and organic selenium increased the activity of antioxidant enzyme systems in BeWo, JEG-3 and Swan-71 and these cells were protected
from oxidative insult, displaying an enhanced level of viability.
Cell viability in these experiments was determined using both the MTT and
Reszaurin end point assays. Both assays showed a dose dependent decrease in the
cellular activity in BeWo, JEG-3 and Swan-71 treated with increasing concentrations
of Rotenone and Antimycin. However there appeared to be a difference in the amount
of Rotenone required to inhibit viability when assessed with MTT (10-100µM) as
compared to resazurin (100-800nM). Reszaurin is a fluorescent based assay which
offers greater sensitivity so this could explain this differential. Prior incubation with
NaSe or SeMet was able to protect trophoblast cells from oxidative stress in both
assays. Selenium supplementation of JEG-3 and BeWo cell lines resulted in protection
against oxidative stress induced at Antimycin concentrations 120-320µM, with
protection against Antimycin concentrations as low as 80µM observed in Swan-71
cells. At 100nM NaSe and 500nM SeMet, a substantial reduction in ROS was seen,
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Selenium and mitochondrial oxidative stress
Chapter 4
that may have prevented the cells from undergoing cell death through apoptosis and
necrosis. The rationale behind this protection is supported by selenium
supplementation, which up-regulates anti-oxidant enzymes, GPx and Thx-Red that
may decrease oxidative stress. These studies strongly suggest that mitochondrial
oxidative stress can be restricted with a simple micronutrient supplement, which
clearly shows a link between selenium, the anti-oxidant proteins GPx and Thx-Red and
mitochondrial oxidative stress. The Swan-71 cells are thought to be an truer
representation of trophoblat cells in- vivo. Therefore, they were our cell model of
choice, In most experiments we vaildated the observations in both JEG-3 and BeWo
but often not to the n values performed with Swan-71 (n=6 in Figure 4.3). Chapter 4
contains 26 individual graphs so we thought that in some instances it was appropriate
to state that a similar observation was made in the other cell types without presenting
all the data.
These observations demonstrate that selenium supplementation may be of
benefit for women who experience pre-eclampsia. There have been several reports
highlighting that pre-eclamptic mothers may be seleno-deficient and selenium intake
may be important in the development of pre-eclampsia. In one report, researchers
measured the selenium concentrations in toenail clippings of pregnant women and
found a significant correlation between pre-eclampsia and decreased selenium status
prior to the onset of disease (Rayman et al., 2003). Thus, selenium concentration can
be determined before the development of symptoms and even before pregnancy, as
toenails are laid down from 3 to 4 months before clipping. This has been supported by
animal studies which also suggest the importance of selenium in pregnancy (Nogales
et al., 2013). Work from this laboratory showed that pregnant rats when fed a selenium
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Selenium and mitochondrial oxidative stress
Chapter 4
depleted diet developed the symptoms of pre-eclampsia (Vanderlelie et al., 2004). This
is an interesting model and one of only a few rodent models of this disease and it
emphasises the importance of this micronutrient in normal reproductive function.
During the third trimester of gestation, increased oxidative stress can be
observed in pregnancies complicated by pre-eclampsia and Intrauterine Growth Factor
(IUGR) (Ebuehi et al., 2003). This causes increased levels of oxidised lipids, proteins
and DNA present in placental tissues and in the circulation of pre-eclamptic mothers
(Hubel, 1999). There is also a significant increase in circulating microparticles,
basement membrane fragments and fetal DNA, which is indicative of increased
trophoblast turnover in association with increased trophoblast apoptosis (Mezian et al.,
2006).
During
pre-eclampsia,
increased
debris
release,
including syncytial
microparticles, causes endothelial cell activation and initiates the onset of
inflammation and immune responses in the mother (Redman et al., 2000, Redman,
2011). There is also evidence of an increase in endothelial adhesion molecules
observed in pre-eclamptic women (Powe et al., 2011) suggesting changes in the
interaction of endothelial cells and circulating immune cells. In addition, endothelial
cells are activated and release various cytokines which are abnormally increased in
pre-eclamptic placentas. To limit this cascade, one potential therapy would be to
increase the expression of key components of anti-oxidant systems which could limit
the damaging effects of oxidative stress.
There have been several reports, which suggest that decreased levels of GPx
and Thx-Red lead to oxidative stress. GPx and Thx-Red are the endogenous antioxidant enzyme systems, which contain selenocysteine (Mistry et al., 2008). As shown
in Chapter 3 small quantities of selenium when added to cell culture medium
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Selenium and mitochondrial oxidative stress
Chapter 4
significantly increase the cellular expression and activity of GPx and Thx-Red.
Placental cells treated this way are more resistant to oxidative stress and contained less
reactive oxygen species as measured by the DCFDA assay. It was observed that at
320µM Antimycin and 100µM Rotenone resulted in maximum reactive oxygen
species production and this affected cell viability as determined with both the MTT
and Resazurin assays. The next question is could endogenous inhibitors of the electron
transport chain such as Antimycin and Rotenone generate mitochondrial oxidative
stress leading to apoptosis or necrosis and could this be reduced with selenium
supplementation, which has been investigated in Chapter 5 of this thesis.
94
CHAPTER 5
SELENIUM
SUPPLEMENTATION AND
PROTECTION FROM APOPTOSIS
Selenium supplementation and protection from apoptosis
5.1
Chapter 5
ABSTRACT
In Chapter 4 of this thesis, it was clearly demonstrated that selenium
supplementation was able to protect trophoblast-like cells from mitochondrial based
oxidative stress induced by Rotenone and Antimycin. In this chapter, we investigated
whether Rotenone is able to induce apoptosis in trophoblast-like cells and the capacity
of selenium treatment to protect against apoptosis. BeWo, JEG-3 and Swan-71 cells
were cultured with 100nM NaSe for 24 hours and subsequently exposed to Rotenone
treatment for 4 hours. Apoptosis was assessed by down regulation of Bcl-2 and via
Annexin V binding and flow cytometry. Rotenone caused trophoblast cells apoptosis
as evidenced by decreased amounts of Bcl-2, a result validated by increased Annexin
V binding. In both assays of apoptosis, selenium supplementation was able to prevent
apoptosis and protect trophoblast cells. In conclusion, this data suggest that selenium is
able to protect trophoblast cells from undergoing apoptosis in response to oxidative
stress generated through inhibition of the electron transport chain.
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Selenium supplementation and protection from apoptosis
5.2
Chapter 5
INTRODUCTION
The term apoptosis describes a process of programmed cell death which occurs
under stressful physiological conditions and selectively removes cells whose function
has been compromised (Tsujimoto, 1998). Apoptosis can be induced by various
stimuli including oxidants, radiation and other chemotherapeutic agents (Matés et al.,
2000). An apoptotic cell undergoes various morphological and biochemical changes
which include; cell shrinkage, nuclear fragmentation and chromatin condensation
(Reed, 2000). Ultimately apoptosis activates the caspase cascade of proteolytic
enzymes leading to cell death and turnover.
Reactive Oxygen Species (ROS) and mitochondria play a critical role in
apoptosis. Mitochondria are the major source of ROS that result in oxidative stress and
lead to the release of Cytochrome c from the inner mitochondrial membrane,
subsequent caspase activation and cell death (Simon et al., 2000). Control and
regulation of apoptosis is governed by Bcl-2 proteins that control the mitochondrial
permeability transition pore, an important step in the intrinsic activation of apoptosis
(Elmore, 2007, Kalogeris et al., 2014). In Chapter 4 of this thesis, it was demonstrated
that inhibition of the electron transport chain through the action of Rotenone and
Antimycin resulted in the generation of free radicals and induced oxidative stress.
Selenium supplementation was also found to maintain oxidative balance in trophoblast
cells and protect against endogenous ROS. In this chapter, we investigated the capacity
of Rotenone to induce apoptosis in trophoblast-like cells by examining Bcl-2
expression and the effect of selenium supplementation on the modulation of this
process.
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Chapter 5
AIMS
The specific aims of this study were:
1 To measure apoptosis with Bcl-2 expression by western blotting in trophoblastlike cells with Rotenone treatment and selenium supplementation.
2 To measure the apoptosis with Annexin V-FITC staining by Flow cytometry in
trophoblast-like cells with Rotenone treatment and selenium supplementation.
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Chapter 5
MATERIALS AND METHODS
5.4.1 Cell Culture
BeWo, JEG-3 and Swan-71 cell lines were cultured at 37°C in 5% CO2 and
21% O2 in DMEM containing 10% fetal bovine serum and 500U/mL penicillin–
streptomycin (GIBCO Life Technologies, Australia). Cells were trypsinized and
subcultured as detailed in section 2.1.1.
5.4.2 Selenium Treatment
Trophoblast-like cells were seeded in 6 well plates and adjusted to a cell
number of 150000 cells per well and 2mL of DMEM was added in each well. The cells
were supplemented with 100nM NaSe for 24 hours at 37°C in 5% CO2 to allow for cell
attachment.
5.4.3 Rotenone Treatment
BeWo, JEG-3 and Swan-71 cells were treated with increasing concentrations of
Rotenone at 10μM, 20μM, 50μM and 100μM. The cells were incubated for 4 hours at
37°C in 5% CO2 before analysis for apoptosis.
5.4.4 Western Blotting
Following selenium treatments cells were exposed to Rotenone concentrations
at 10μM, 20μM, 50μM and 100μM for 4 hours at 37°C in 5% CO2 to generate
apoptosis. Cell extracts were generated as previously described section 2.1.3. Protein
estimations were done as detailed in section 2.2.1. Western blotting was undertaken as
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described in detailed in section 2.7. The membranes were probed with a primary
antibody of rabbit polyclonal Bcl-2 antibody (Cell Signaling Technology, Australia) at
a dilution of 1:1000 in TBS with 5% BSA and 0.1%Tween-20 overnight at 4ºC on a
shaker. This was followed by subsequent washes of TBST as mentioned in section 2.7.
The membranes were incubated with horseradish peroxidase-conjugated goat antirabbit secondary IgG antibody at a dilution of 1:5000 (Santa Cruz Biotechnology,
USA) in 5% BSA and 0.1% Tween-20 for 1 hour at 37°C on a shaker. The processing
of the blots was done as mentioned in methods section 2.67and densitometry analysis
was performed.
5.4.5 Flow Cytometry
Following treatments and incubations as described earlier the cells were
trypsinized and collected. Cells were spun at 1100 rpm for 5 minutes, the supernatant
was discarded and the cell pellet was washed twice with cold PBS. The cells were
suspended in 100μL 1x Annexin V binding buffer (BD Biosciences) plus 3μL of
Annexin V. Fluorescein isothiocyanate (FITC) (BD Biosciences) and 1μL of
100μg/mL propidium iodide. Cells were gently vortexed and incubated for 15 minutes
at room temperature in the dark. 400μL of 1x binding buffer was added in each sample
and analysed by BDTM Flow cytometer (BD Biosciences, California, USA).
5.4.6 Statistical Analysis
All the data was analysed using Graph Pad Prism, version 6.0 with p<0.05
considered significant in all tests. ANOVA test was used to analyse the differences
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Selenium supplementation and protection from apoptosis
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between control and treatment groups. Post-hoc Tukey’s test was performed after
performing ANAOVA test as described in section 2.8.1.
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Chapter 5
RESULTS
5.5.1 Expression of Bcl-2 induced by Rotenone and effect of NaSe
supplementation on apoptosis in trophoblast cells
BeWo, JEG-3 and Swan-71 cells were grown in DMEM, with and without
100nM NaSe for 24 hours and treated with Rotenone at 10μM, 20μM, 50μM and
100μM for 4 hours. Western blot analysis for apoptotic proteins was performed on cell
extracts to study the effect of Rotenone and the impact of NaSe in preventing cells
from undergoing apoptosis. The levels of expression of Bcl-2 in trophoblast cells post
treatment is illustrated in Figures 5.1 A, B and C.
Figure 5.1 Bcl-2 expression in BeWo (A), JEG-3 (B) and Swan-71 (C) cells treated
with selenium and later exposed to Rotenone. Images show the
representative western blot bands; CNTL=control.
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It was observed that at 100μM Rotenone, the expression of Bcl-2 in BeWo,
JEG-3 and Swan-71 cells was significantly reduced by 0.39, 0.37 and 0.10 fold
respectively as measured through densitometry analysis (Figure 5.2 A, B and C).
However, after supplementing with 100nM NaSe, the Bcl-2 expression was seen to be
up-regulated to control levels of 0.87 (P<0.001), 0.83 (P<0.01) and 0.48 (P<0.05) fold
in BeWo, JEG-3 and Swan-71 cells respectively (Figure 5.2 A, B and C). Similar
observations in Bcl-2 expression were made in BeWo and JEG-3 cells when
supplemented with 20μM and 50μM Rotenone.
Figure 5.2 Densitometry of western blots quantitating the expression levels of Bcl-2 in
BeWo (A), JEG-3 (B) and Swan-71 (C) cells normalised to actin
expression. Values are presented as mean ± standard deviation (SD);
CNTL=control (*p<0.05, p** <0.01, ***p<0.001, n=4).
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5.5.2 Quantification of apoptosis in trophoblast cells using Annexin V FITC
Annexin V staining was used to detect apoptosis in BeWo and JEG-3 cells via
flow cytometry. BeWo and JEG-3 cells were seeded in 6 well plates in DMEM and
supplemented with 100nM NaSe for 24 hours. Cells were subsequently treated with
100μM Rotenone for 4 hours. Apoptotic cells exclude cell viability dyes such as
Propidium Iodide (PI) whereas the cells which are undergoing necrosis do not exclude
PI. The lower left quadrant (Q4) in the (Figures 5.3 A and B) shows viable live cells,
which exclude PI and are negative for Annexin V FITC binding. The upper right
quadrant represents the non-viable cells or necrotic cells, which are positive for
Annexin V FITC binding and showing PI uptake (Q2). The lower right quadrant (Q3)
represents the percentage of apoptotic cells Annexin V FITC positive and PI negative,
demonstrating Annexin V binding and cytoplasmic membrane integrity.
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Figure 5.3 Annexin V FITC staining in untreated BeWo cells (A), when cells are
exposed to 100μM Rotenone (B) and cells treated in combination with
selenium and Rotenone (C). Annexin V FITC staining in untreated JEG-3
cells (D), when cells are exposed to 100μM Rotenone (E) and cells treated
in combination with selenium and Rotenone (F). Representative graphs of
BeWo and JEG-3 showing the percentage of apoptotic cells. Values are
presented as mean ± standard deviation (SD). (*p<0.05, p** <0.01, n=4).
The apoptotic cell population in BeWo cells when treated with 100μM
Rotenone was 58.72 ± 9.96% (P<0.05). Supplementation with selenium not only
improved the viability of cells (Figure 5.3 C) but also significantly lowered the
percentage of apoptotic cells to 31.35 ± 3.08% (P<0.001). In JEG-3 cells, the total
population of cells undergoing apoptosis after Rotenone exposure was 65.50 ± 4.76%
(Figure 5.3 E). Supplementation with NaSe increased the percentage of live cells and
reduced the effect of apoptosis (40.80 ± 0.984%, Figure 5.3 F) (P<0.001).
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Chapter 5
DISCUSSION
Apoptosis is defined as a process of programmed cell death, which occurs
under physiological conditions. It is critical for normal cell turnover and plays an
important role in embryonic development and tissue homeostasis (Elmore, 2007).
Dysregulation of apoptosis can lead to diseases such as cancer, Parkinson’s disease,
heart failure and other age related diseases (MacFarlane et al., 2004). Apoptosis is a
complex process that involves a cascade of molecular events, with the final death
phase of the apoptosis pathway being initiated by the cleavage of caspase-3, resulting
in DNA damage and degradation of cytoskeletal and nuclear proteins (Reed, 2000).
Subsequently, there is formation of apoptotic bodies and finally uptake by phagocytic
cells that remove the cell debris in a non-inflammatory manner (Martinvalet et al.,
2005).
Mitochondria are the major site for ROS generation and contribute to oxidative
stress and damage to cells and tissues (Marchi et al., 2012). There is extensive
evidence suggesting that ROS drive apoptosis through an intrinsic mechanism of
activation (Scaffidi et al., 1998). ROS generation causes depolarisation of the inner
mitochondrial membrane and results in the opening of the mitochondrial permeability
transition pore and release of Cytochrome c which activates the caspase cascade
leading to cell death (Kalogeris et al., 2014). In Chapter 4 of this thesis it was observed
that inhibitors of the electron transport chain like Antimycin and Rotenone could cause
ROS production and oxidative damage in trophoblast cells via a mechanism of electron
leakage and the formation of superoxide. Selenium supplementation protected these
cells and also improved the cellular viability. In this chapter, we aimed to extend these
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studies by inducing apoptosis with Rotenone treatment in cells which that had been
cultured with and without NaSe.
Apoptosis was measured by Bcl-2 protein through western blotting. Bcl-2
protein is an apoptosis regulator and controls the mitochondrial membrane
permeability transition pore (MPTP) (Brunelle et al., 2009). The anti-apoptotic Bcl-2
protein prevents apoptosis by stabilising the pore, inhibiting the release of Cytochrome
c from the mitochondria, thereby preventing the activation of caspases (MacManus et
al., 1997). When BeWo, JEG-3 and Swan-71 cells were treated with 10μM, 20μM,
50μM and 100μM concentrations of Rotenone for 4 hours, it was observed that there
was a significant decrease in Bcl-2 protein, corresponding to an increase in apoptosis
in a dose-dependent manner. However, when the cells were supplemented with 100nM
NaSe for 24 hours, the level of Bcl-2 expression did not decrease providing protection
from apoptosis in all three trophoblast-like cell lines.
Similar observations were made when Annexin-V was used to detect apoptosis.
Annexin-V binds to externalised phosphotidyl-serine which is indicative of a cell
undergoing apoptosis. Flow cytometry data showed that when cells were treated with
100μM Rotenone, there was a loss of membrane integrity and increased staining with
Annexin V FITC, thereby indicative of induction of apoptosis. However, cells were
able to maintain their membrane integrity upon selenium treatment and thereby
preventing apoptosis in response to intrinsic activation through ROS production.
Presumably this is mediated through the increased expression of selenoproteins GPx
and Thx-Red which are important anti-oxidants that limit the detrimental effects of
ROS.
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Apoptosis is important for placental development and has also been shown to
play a role in supporting maternal immune tolerance expressed by trophoblast cells
(Straszewski et al., 2005). Apoptosis can be observed in trophoblast cells and in
placental tissues throughout pregnancy (Smith et al., 1997) and plays an active role in
the shedding of placental material during normal cell turnover. Apoptosis is essential
for villous trophoblast throughout pregnancy and is crucial for placental invasion,
cytotrophoblast fusion and syncytiotrophoblast function. Dysfunction of placental
function by generation of ROS and hypoxia leads to significant increase in placental
apoptosis (Sharp et al., 2010). In complications of pregnancy like pre-eclampsia there
is an increase in the rate of apoptosis observed suggesting that alterations in the
regulation of trophoblast apoptosis may contribute to the pathophysiology of this
disease (Allaire et al., 2000).
In conclusion, these data suggest that trophoblast cells are able to induce
apoptosis when stimulated by Rotenone to generate a mitochondrial specific oxidative
insult. Rotenone caused cytotoxicity and oxidative stress in a concentration dependent
manner in trophoblast-like cells. This effect was limited when increasing
concentrations of sodium selenite was present. Selenium supplementation could have
the potential to lower or combat the mechanism of apoptosis by reducing ROS
production in trophoblast cells. In pregnancy it is important to control the production
of placental debris which is generated through apoptosis and shed in to the maternal
circulation as this can activate the maternal endothelium and lead to disease such as
pre-eclampsia.
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CHAPTER 6
SELENIUM INDUCED
MITOCHONDRIAL BIOGENESIS IN
TROPHOBLAST
PUBLICATIONS ARISING FROM THIS CHAPTER
Khera A, Dong LF, Holland O, Vanderlelie J, Pasdar EA, Neuzil J, Perkins AV.
(2015) Selenium supplementation induces mitochondrial biogenesis in trophoblasts.
Placenta 36: 863-9.
Selenium induced mitochondrial biogenesis in trophoblast
6.1
Chapter 6
ABSTRACT
It was previously demonstrated that selenium is able protect trophoblast cells
through the induction of anti-oxidant enzymes GPx and Thx-Red. The data presented
in Chapters 4 and 5 suggest that this protective mechanism is due to reduced levels of
reactive oxygen species, increased cell viability and protection form apoptosis. This
chapter examines how selenium increases the mitochondrial function of trophoblast
cells and activates mitochondrial biogenesis through various pathways including
activation of transcription factors such as Nuclear Respiratory Factor-1 (NRF-1) and
Peroxisome
Proliferator-Activated
Receptor
Coactivator-1alpha
(PGC-1α).
Trophoblast cells (BeWo, JEG-3 and Swan-71) were treated with Sodium Selenite
(100nM) or Selenomethionine (500nM) for 24 hours. Cellular respiration was then
measured
using
an
Oxygraph-2k
(Oroboros)
high
respirometry
chamber.
Mitochondrial number was determined with qPCR and citrate synthase assays. The
expression of the mitochondrial biogenesis proteins Selenoprotein H, PGC-1α and
NRF-1 was examined by western blotting. Mitochondrial respiration and oxygen
consumption was significantly higher in cells treated with NaSe as compared to nontreated cells. qPCR and citrate synthase assay data revealed that mitochondrial content
was higher in cells treated with selenium. The expression of mitochondrial biogenesis
proteins Selenoprotein H, PGC-1α and NRF-1 was up-regulated as determined by
western blotting. These results give special attention to the importance of selenium in
ensuring mitochondrial regeneration and this may play an additional role in protecting
trophoblast cells from oxidative stress. Considering that selenium deficiency is
associated with complications of pregnancy, supplementation may provide a useful
therapy.
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Selenium induced mitochondrial biogenesis in trophoblast
6.2
Chapter 6
INTRODUCTION
Development of oxidative stress is linked to the generation of ROS in
mitochondria and cell survival is solely dependent on how mitochondria are able to
counteract the deleterious actions of ROS via anti-oxidant expression. Any disruption
to the flow of electrons through the electron transport chain results in the production of
partially reduced forms of oxygen and nitrogen, which constitute the reactive oxygen
and nitrogen species (RONS). In this context, it was previously demonstrated that
selenium supplementation was an effective means to enhance the anti-oxidant capacity
and thereby increase mitochondrial ability to resist oxidative stress in placental
trophoblast cells (Chapter 4 of this thesis, Khera et al., 2013, Watson et al., 2012).
Oxidatively stressed mitochondria are able to return to normal function via
anti-oxidant defences and through a process of fusion and fission (Youle et al., 2012).
Conversely, the process of autophagy removes mitochondria if they are not able to
recover. If the mitochondrial dysfunction is in excess then there is release of
Cytochrome c, which can lead to apoptosis (Galluzzi et al., 2012). This process of cell
turnover is important in the human placenta where mitochondrial oxidative stress can
drive changes in the shedding of placental debris into the maternal circulation. This
can induce a systemic maternal immune response and endothelial cell activation,
which is responsible for pathophysiology of pre-eclampsia (Poston et al., 2011).
In Chapter 4 of this thesis it was demonstrated that selenium supplementation
protected trophoblast cells from mitochondrial oxidative stress through the upregulation of anti-oxidant enzyme systems. Selenium may also be responsible for
improving mitochondrial function through the regulation of mitochondrial biogenesis
by activation of mitochondrial regeneration pathways through a recently described
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novel selenoprotein, Selenoprotein H (Sel H) which further activates mitochondrial
regeneration pathways (Mehta et al., 2013). In this chapter we investigate the effect of
selenium on mitochondrial respiration and to determine if the improvement in
mitochondrial function was due to an increase in mitochondrial content, and if this
might be regulated through Sel H.
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Chapter 6
AIMS
The specific aims of this study were:
1 To measure mitochondrial function/respiration of trophoblast-like cell lines
supplemented with selenium, using Oxygraph-2k Oroboros high respirometry
chamber.
2 To determine the effect of selenium supplementation on mitochondrial content
of trophoblast-like cells using qPCR and citrate synthase assay.
3 To investigate the expression of mitochondrial biogenesis proteins Sel H, PGC1α and NRF-1 in trophoblast-like cells supplemented with selenium by western
blotting.
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6.4
Chapter 6
MATERIALS AND METHODS
6.4.1 Cell Culture
BeWo, JEG-3 and Swan-71 cell lines were grown in DMEM (GIBCO Life
Technologies, Australia) containing 10% fetal bovine serum and 500U/mL penicillin–
streptomycin (GIBCO Life Technologies, Australia) in 5% CO2 and 21% O2 at 37°C.
Cells were subcultured as described in section 2.1.1. Cells were treated with 100nM
Sodium Selenite (Sigma, Australia) or 500nM Selenomethionine (Sigma, Australia)
for 24 hours.
6.4.2 Measurement of mitochondrial respiration in trophoblast –like cell lines
Mitochondrial respiration was measured in an Oxygraph-2k (Oroboros
Instruments, Innsbruck, Austria) high respirometry chamber operated at 37°C.
Trophoblast cell lines cultured with or without 100nM Sodium Selenite (NaSe) for 24
hours were tested for both ROUTINE respiration and respiration of individual
mitochondrial complexes at a density of 1 million cells/mL in 2.0mL Oxygraph-2k
chambers running in parallel.
For the first protocol, (substrate-uncoupler-inhibitor titrations; SUIT protocol
one), a suspension of 1 million cells per mL in DMEM was added to the Oxygraph-2k
chambers and examination of cellular respiration was conducted under four conditions:
(1) ROUTINE respiratory state with the physiological substrates present in the culture
medium, (2) LEAK respiratory state when ATP synthase is inhibited by Oligomycin
(4mg/mL), (3) electron transfer system (ETS) capacity at non-coupled respiration after
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the addition of carbonyl cyanide p-trifluoro-methoxyphenyl hydrazone (FCCP; 1mM),
and (4) residual oxygen consumption (ROX) after the addition of Rotenone (1μM).
For the second protocol, (SUIT protocol two), a suspension of 1 million cells
per mL in mitochondrial respiration medium MiRO5 (110mM sucrose, 0.5mM EDTA,
3.0mM MgCl2, 60mM K-lactobionate, 10mM KH2PO4, 20mM HEPES, 1.0g/L BSA
and Taurine 20mM pH 7.1) was added to the O2k chamber. Digitonin at a
concentration of 6mg/mL was added to permiabilised the cells. Pyruvate (5mM),
glutamate (10mM) and malate (2mM) were added to measure complex I (CI)-mediated
LEAK respiration, then oxidative phosphorylation (OXPHOS) was stimulated by the
addition of ADP (1-5mM). To test the integrity of the outer mitochondrial membrane,
Cytochrome c at a concentration of 10μM was added. Succinate (10mM) was then
used to stimulate OXPHOS through complex II (CII), followed by titrations of FCCP
(1mM) to investigate uncoupling of the mitochondria. The addition of Rotenone (1μM)
and Antimycin A (2.5μM) inhibited CI and Complex III (CIII), respectively.
6.4.3 DNA Extraction
DNA was extracted from Swan-71 cells using the Genomic DNA Purification
Kit (Thermo Scientific, Australia) following the manufacture’s protocol. 0.8 x 106 cells
were collected by centrifugation, resuspended in 400μL of lysis buffer and incubated at
65°C for 5 minutes. Chloroform (600μL) was added immediately to the sample and
emulsified by inverting 3-5 times. The sample was centrifuged for 2 min at 10000 rpm
and the upper aqueous layer phase containing DNA was transferred to a new tube. The
precipitation solution was added and the sample was gently mixed by several
inversions at room temperature followed by centrifugation at 10000rpm for 2 min. The
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Selenium induced mitochondrial biogenesis in trophoblast
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supernatant was removed completely and DNA pellet was dissolved in 100μL of NaCl
solution by gentle vortexing. Cold ethanol (300μL) was added to precipitate the DNA
for 10 min at -20°C. The sample was centrifuged at 10000rpm for 3-4 min to remove
the ethanol and collect the DNA pellet. The pellet was suspended in 50μL of sterile
deionized water by gentle vortexing and the DNA concentration was measured on the
2000c Nanodrop (Thermo Scientific, Australia) according to the manufacturer’s
protocol.
6.4.4 Real time Polymerase Chain Reaction (RT-qPCR) for determining
mitochondrial DNA copy number
Real-time quantitative PCR (RT-qPCR) was performed using 25ng/μL of total
DNA, as described in section 2.5. The DNA was combined with 10μM of forward and
reverse primers (Table 6.1) and assessed with 5x Eva Green qPCR SuperMix (Solis
BioDyne, Australia) on an Eco qPCR System (Illumina, Australia). Mitochondrial
DNA content in cells was time PCR calculated by the comparative CT method. The
comparative CT method calculates a ratio, rather than an absolute amount. The quantity
of four mitochondrial genes (Table 6.1) was normalised to a single-copy nuclear gene
(nuclear receptor coactivator 3) (Table 6.1) to give a ratio of the number of
mitochondrial genomes per nuclear genome. An internal assay reference gene (β-actin)
was also included. Relative quantification values were calculated from cycle threshold
values (CT) using 2−ΔΔCT; where ΔΔCT compares each mitochondrial gene with the
nuclear gene and the final calculation expresses this as a ratio change based on
amplification (Schmittgen et al., 2008).
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Selenium induced mitochondrial biogenesis in trophoblast
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Table 6.1. Real time PCR primers
Genes
Target
Forward Primer
Reverse Primer
MTRT1
mtDNA
CAC CCA AGA ACA GGG TTT GT
TGG CCA TGG GTA TGT TGT TA
MTRT2
mtDNA
TCC TCC TAT CCC TCA ACC CC
CAC AAT CTG ATG TTT TGG TTA
AAC
MTRT3
mtDNA
CAT CTG GTT CCT ACT TCA
TGA GTG GTT AAT AGG GTG ATA
GGG
GA
CAT TTT GGT TCT CAG GGT TTG
AGG CAG CTC GTA GCT CTT CTC
MTRT4
mtDNA
ATG GCC CAC CAT AAT TAC CC
MTBA
nDNA
AGC GGG AAA TCG TGC GTG AC
mtDNA= mitochondrial DNA; nDNA= nuclear DNA.
6.4.5 Western Blotting
Trophoblast cell lines BeWo, JEG-3 and Swan-71 were cultured in 6-well
plates (Grenier Bio-One Cellstar) at a density of 0.15 x 106 cells and supplemented
with NaSe at 100nM and 500nM SeMet for 24 hours in 5% CO2 at 37°C incubator.
Cell extracts and protein estimations were done as outlined in section 2.2.2. Western
blot using antibodies for Sel H (Santa Cruz Biotechnology, USA), PGC-1α (Cell
Signaling Technology, Australia), NRF-1 (Cell Signaling Technology, Australia), and
β-actin (Cell Signaling Technology, Australia) was carried out as described in section
2.7.
6.4.6 Citrate Synthase Assay
Citrate synthase is the initial enzyme of the tricarboxylic acid (TCA) cycle
catalysing the reaction between acetyl coenzyme A (acetyl CoA) and oxaloacetate to
form citrate. This enzyme is a marker of the mitochondrial matrix and can be used to
assay mitochondrial content. Mitochondrial citrate synthase activity was measured by
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Selenium induced mitochondrial biogenesis in trophoblast
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the reduction of 5, 5′-dithiobis-(2-nitrobenzoic acid) (DTNP) by citrate synthase and
using the coupled reaction with acetyl-CoA and oxaloacetate. Swan-71 cells treated
with ± 100nM NaSe were seeded in a 12- well plate (Grenier Bio-One CellStar,
Australia) at a density of 80000 cells/well and incubated for 24 h at 37°C. 10μg of
protein lysate was added to 10mM DTNB and 30mM acetyl coenzyme A and 10mM
oxaloacetate was added to initiate the reaction. Citrate synthase enzyme (Sigma,
Australia) was used as positive control. The change in absorbance of DTNB was
measured spectrophotometrically in a kinetic program for 90 seconds at 412nm at
30°C on an Infinite M200 Pro Tecan machine and the activity was calculated as
described by (Eigentler et al., 2012).
6.4.7 Statistical Analysis
All the data are presented as mean ± standard deviation. Statistical analysis was
performed using Graph Pad, PRISM version 6 for Windows (GraphPad Software, San
Diego California USA, www.graphpad.com). A Grubbs’ test was used to determine
significant outlier values. Kruskal-Wallis test with Dunn's multiple comparison tests
was used in Figure 6.1 and Figure 6.2 to analyse the difference between the treatments
and a two-tailed Wilcoxon matched-pairs signed rank test in Figure 6.3 and Figure 6.4.
P values >0.05 were considered significant. Each result is a minimum of five
individual experiments.
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Selenium induced mitochondrial biogenesis in trophoblast
6.5
Chapter 6
RESULTS
6.5.1 Selenium enhances mitochondrial respiration in trophoblast -like cell lines
Oxygen consumption in BeWo, JEG-3 and Swan-71 cells was measured in
Oxygraph- 2k (Oroboros Instruments, Innsbruck, Austria) high respirometry chamber.
The respiration of intact BeWo, JEG-3 and Swan-71 cells was investigated initially
and subsequently Swan-71 cells were permiabilised to determine mitochondrial
complex-specific fluxes. Mitochondrial respiration was significantly higher when cells
were treated with 100nM NaSe for 24 hours as compared to untreated controls in
nearly all states investigated, (Figure 6.1) indicating increased respiratory capacity
with selenium supplementation in trophoblast-like cell lines.
In intact cells, both ROUTINE respiration and electron transfer system (ETS)
capacity were significantly increased in BeWo and Swan-71 cell lines when
supplemented with selenium as compared to untreated controls (Figure 6.1 A). The
ROUTINE respiration in Swan-71cells selenium treated cells was 38.54 ± 5.28 as
compared to 19.66 ± 4.62 in the cells without selenium supplementation (P<0.05). The
ETS capacity in selenium treated cells was also significantly higher than non-treated
cells; 68.28 ± 14.49 vs 26.34 ± 2.52, P<0.05 JEG-3 and BeWo cells were also tested
and showed similar effects. The ETS capacity in JEG-3 cells when they were
supplemented with NaSe was 88.92 ± 12.95 as compared to non-selenium treated cells
55.70 ± 24.75 (P<0.05). The ROUTINE respiration and ETS capacity in BeWo cells
was 31.26 ± 5.69, 40.19 ± 17.97 respectively, however when supplemented with
selenium both ROUTINE respiration and ETS capacity significantly boosted 52.60 ±
10.59, 66.23 ± 7.83 respectively. Thus, the selenium treated mitochondria functioned
at a higher capacity under normal conditions, and also demonstrated greater reserves of
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Selenium induced mitochondrial biogenesis in trophoblast
Chapter 6
respiratory capacity in all three trophoblast cell lines. Further, in permeablised Swan71 cells it was found that the increased respiratory capacity in selenium treated cells
was largely due to increased oxidative phosphorylation (OXPHOS) through a
significant increase in complex I and a smaller non-significant increase in cmplex II
(Figure 6.1 B).
Figure 6.1 (A) Intact cells; a representative oxygraph trace of oxygen flux relative to
cell number in non-selenium (light grey) and selenium-treated (dark grey)
cells. Mitochondrial ROUTINE and ETS respiration rates observed in
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Selenium induced mitochondrial biogenesis in trophoblast
Chapter 6
BeWo, JEG-3 and Swan-71 cells, respectively. (B) Permiabilised Swan-71
cells; a representative Oxygraph trace of oxygen flux relative to cell
number in non-selenium (light grey) and selenium-treated (dark grey).
Oxidative phosphorylation (OXPHOS) through complex I, complex II and
ETS capacity in permiabilised Swan-71 cells. Data was acquired from five
individual experiments for intact Swan-71, eight individual experiments
for intact JEG-3 , six individual experiments for intact BeWo and seven
individual experiments for permiabilised Swan-71 cells ; Values are means
± S.D (*p<0.05).
6.5.2 Selenium increases mitochondrial content in trophoblast-like cells
To determine if the observed increase in mitochondrial respiration was due to
improved mitochondrial function or increased mitochondrial content, mitochondrial
content was measured in Swan-71 cells. There was a significant increase in the
mitochondrial content when Swan-71 cells were supplemented with both sources of
selenium, NaSe (Figure, 6.2A) and SeMet (Figure 6.2 B), as measured by the ratio of
mtDNA to nDNA. All four mitochondrial genes investigated MTRT1, MTRT2, MTRT3
and MTRT4 (Figure 6.2 A and B) were significantly increased in the cells
supplemented with selenium (NaSe or SeMet), as compared to the cells grown in nonsupplemented media when normalised to the referring nuclear gene, β-actin.
Combining all mitochondrial genes it was observed that NaSe increased the fold
difference in expression by 2.03 ± 0.14 and SeMet increased the fold difference in
expression by 2.14 ± 0.12. To confirm the results observed with mtDNA/nDNA ratios,
a citrate synthase assay was performed on Swan-71 cells treated with NaSe, compared
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to untreated controls. The selenium treatment significantly increased citrate synthase
activity to 176.91 ± 14.70 pmol/mg of protein as compared to the activity in the nontreated cells, 131.12 ± 8.35. pmol/mg of protein, an increase of 1.35 fold (Figure 6.2
C).
Figure 6.2 (A) Mitochondrial content is increased in Swan-71 cells lines
Supplemented with selenium. (A) Mitochondrial DNA copy number
(mtDNA/nDNA ratio), in Swan-71 cells treated with 100nM NaSe
compared to untreated controls and (B) 500nM SeMet compared to
untreated controls. (C) Citrate synthase activity in Swan-71 cells treated
with 100nM NaSe compared to untreated controls. Data was acquired
from six individual experiments. Values are means ±S.D. (*p<0.05).
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6.5.3 Selenium up-regulates mitochondrial biogenesis markers
To investigate if selenium supplementation could influence the ability of
trophoblast cells to generate new mitochondria, the mitochondrial biogenesis markers
Sel H, PGC-1α and NRF-1 were investigated in Swan71, BeWo and JEG-3 cells. A
significant increase in the expression of Sel H was observed in all trophoblast-like cell
lines when cells were supplemented with either NaSe (Figure 6.3 A) or SeMet (Figure
6.3 B); with fold increases of 1.84–3.65. The expression of PGC-1α and NRF-1 were
also significantly increased in selenium treated Swan-71 cells; with fold increases
1.99–5.44, (Figure 6.4 A, B, C and D).
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Figure 6.3 Sel H expression in BeWo, JEG-3 and Swan-71 cells treated with selenium
100nM NaSe (A) and 500nM SeMet (B) compared to controls. Images
show representative western blot bands. Plots show expression levels of
Sel H normalised to actin expression (protein content control), with
untreated control samples then normalised to one to give relative
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expression of Sel H in untreated control and selenium supplemented
samples. Data represents mean ± S.D. and were collected from 6 different
individual experiments; CNTL=control (*p<0.05, **p<0.01).
Figure 6.4 PGC-1 α expression in Swan-71 cells treated with selenium 100nM NaSe
(A) and 500nM SeMet (B) compared to controls. Images show
representative western blot bands. NRF-1 expression in Swan-71 cells with
selenium 100nM NaSe (C) and 500nM SeMet (D) compared to controls.
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Plots show expression levels of PGC-1α (A and B) normalised to actin
expression (protein content control), with untreated control samples then
normalised to one to give relative expression of PGC-1α and NRF-1 (C
and D) in untreated control and selenium supplemented samples. Data
represents mean ± S.D. and were collected from 6 different individual
experiments; CNTL=control ;(*p<0.05).
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6.6
Chapter 6
DISCUSSION
Selenium supplementation has been shown to be effective in protecting
trophoblast cells from oxidative stress when applied exogenously (Watson et al.,
2012). In Chapter 4 of this thesis it was reported that selenium supplementation
protects trophoblast- like cells from mitochondrial oxidative stress when generated
endogenously. Selenium enhanced the activity of anti-oxidant selenoproteins GPx and
Thx-Red, thereby diminishing the destructive effects of reactive oxygen species. These
investigations suggest that selenium supplementation has a protective effect on
mitochondrial function through the action of these anti-oxidant enzymes. In this
chapter, we report that selenium not only protects mitochondria from oxidative stress
but also enhances mitochondrial biogenesis and hence mitochondrial content in
trophoblast cells.
Mitochondrial respiratory rate was significantly boosted with the addition of
100nM NaSe for 24 hours in all trophoblast cell lines (BeWo, JEG-3 and Swan-71).
The improved respiratory rate may be due to the increased protection from anti-oxidant
enzymes such as the selenoproteins GPx and Thx-Red, as shown in Chapter 4 of this
thesis, however recent reports suggest that mitochondrial biogenesis is activated by
selenium, thus enhancing the respiratory capacity of cells and tissues (Mehta et al.,
2012). Therefore, with these recent findings in mind, we investigated whether these
molecular mechanisms could be driving mitochondrial biogenesis in placental
trophoblast cells, thereby increasing mitochondrial content and so providing protection
from cellular demise.
Quantitative RT-PCR and citrate synthase assays were utilised to determine the
mitochondrial content in trophoblast cell lines. Mitochondrial content was significantly
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higher when cells supplemented with selenium, as compared by the ratio of
mtDNA/nDNA. As this data is expressed as a ratio of mitochondrial DNA/nuclear
DNA in provides conclusive proof that cells treated with selenium are able to increase
their mitochondrial content and hence respiratory capacity. This is substantiated by the
increase seen in citrate synthase activity in cells supplemented with selenium. This
assay measures the mitochondrial enzyme citrate synthase and is classically used to
estimate mitochondrial content in cells.
The transcriptional factor Sel H was up-regulated post selenium treatment. Sel
H is a recently characterised 14-kDa selenoprotein associated with enhanced antioxidant capacity, cell viability and mitochondrial function (Mendelev et al., 2011,
Mendelev et al., 2009, Morozova et al., 2003, Panee et al., 2007). Sel H has recently
been found to be an upstream regulator of mitochondrial biogenesis through
mitochondrial biogenesis activators peroxisome proliferator-activated receptor γ
coactivator-1α (PGC-1α) and nuclear respiratory factor 1 (NRF-1). The expression of
PGC-1α and NRF-1 were also significantly higher when trophoblast cells were treated
with selenium. Therefore, mitochondrial content and mitochondrial biogenesis
regulators are increase by selenium supplementation in trophoblast cells. The role of
Sel H in mitochondrial biogenesis has been demonstrated in the neuronal HT22 cell
line (Mehta et al., 2013, Mendelev et al., 2011) and skeletal muscle (Bergeron et al.,
2001). These observations from varied cell types strongly suggest that Sel H may have
an important role in mitochondrial biogenesis in many tissues.
The increase in both mitochondrial function (as measured by respirometry) and
mitochondrial content when cells were treated with selenium suggest that the observed
increase in mitochondrial function may be due to mitochondria biogenesis. Hence,
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selenium treatment not only protects from oxidative insult endogenously by the
regulation of anti-oxidant enzyme systems as shown in Chapter 4 of this thesis, but as
the data supports, it is also responsible for the formation of new mitochondria in
trophoblast cells. Hence, selenium treatment not only protects from oxidative insult
endogenously by the regulation of anti-oxidant enzyme systems as shown in Chapter 4
of this thesis, but our data strongly suggests that mitochondrial biogenesis also occurs
as there is increased citrate synthase activity and qPCR shows increased mitochondrial
content. This is a significant observation as it demonstrates selenium supplementation
has the capability to alter the cell metabolism thereby improving mitochondrial
function and energy production by affecting mitochondrial content. The expression
and activity of mitochondrial complexes in the placenta have been shown to be
reduced in pre-eclampsia (Muralimanoharan et al., 2012), although mitochondrial
content may not be affected (Mandò et al., 2014). The results presented here suggest
that supplementation with selenium may be a way to improve mitochondrial function
in the placenta; therefore selenium is a potential therapeutic for the treatment of preeclampsia.
Mitochondria are the key site of ROS generation which is neutralised by antioxidant enzymes present in mitochondrial matrix. The imbalance between ROS and
anti-oxidants results in oxidative stress. Mitochondrial response to oxidative stress is
important to cellular function and survival, and severe oxidative stress will lead to
necrosis or apoptosis. Oxidative insult in tissue leads to extensive death as is the case
in myocardial infarction and stroke (Fulda et al., 2010) and seen in placentae from
severely preeclamptic women (Bernardi et al., 2008, Huppertz et al., 2003). These
results are important in relation to oxidative stress in placenta, which is integral in
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complications of pregnancy such as pre-eclampsia, gestational diabetes and pre-term
labour (Poston et al., 2011). These complications affect 25 % of all births (Jeyabalan,
2013, King, 1998, Madan et al., 2010), leading to significant morbidity and mortality.
The study reported here, suggests that selenium may be beneficial in preventing
placental oxidative stress in these complications of pregnancy.
Several clinical trials have been conducted to test the benefit of selenium
supplementation in preventing complications of pregnancy. (Rayman et al., 2014, Tara
et al., 2010). The SPRINT (Se in PRegnancy INTervention) trial, which has recently
been published, investigated the biochemical markers associated with the development
of pre-eclampsia and found a significant reduction in soluble fms-like tyrosine kinase1 (sFLT-1) in women treated with selenium (Rayman et al., 2014). sFLT-1 is an antiangiogenic factor that implies as a key mediator in the pathophysiology of preeclampsia (Luttun et al., 2003). Hence, these studies indicate an adequate intake of
selenium during pregnancy may be beneficial in ultimate birth outcome. However,
these studies have been conducted on cells in- vitro so similar effects have been
observed in placental tissues which is investigated in Chapter 7 of this thesis.
.
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CHAPTER 7
SELENIUM
SUPPLEMENTATION PROMOTES
MITOCHONDRIAL FUNCTION IN
PLACENTALTISSUES
Selenium supplementation promotes mitochondrial function in placental tissues
Chapter 7
7.1
ABSTRACT
It was previously demonstrated in Chapter 6 of this thesis that selenium is able
to improve mitochondrial respiration and biogenesis in trophoblast-like cells. The
experiments described in this chapter extend these observations to placental tissues and
examines the effect of selenium supplementation on selenoprotein expression and
mitochondrial function. Villous tissues, from term and 1st trimester placental tissues
were treated with 100nM NaSe for 4, 12, 14, 24, 48 and 96 hours. Western blotting
was performed to determine the expression of selenoproteins Glutathione Peroxidase
(GPx) and Thioredoxin-Reductase (Thx-Red) and the mitochondrial biogenesis
proteins Selenoprotein H, PGC-1α and NRF-1. Quantitative real-time PCR was
conducted on placental tissues to measure the mRNA expression of GPx and Thx-Red
and to determine mitochondrial content post selenium supplementation. Mitochondrial
respiration was measured using an Oxygraph-2k (Oroboros) high respirometry
chamber. GPx and Thx-Red protein expression was significantly enhanced in tissues
with selenium treatment, as was the expression of mitochondrial biogenesis proteins
selenoprotein H, PGC-1α and NRF-1. Mitochondrial content in tissues did not change
with selenium supplementation. Mitochondrial respiration at complex IV was
significantly enhanced at 4 hours however this was not seen in Complexes I or II.
There was a time dependent effect, with significance lost at longer treatment times.
These results suggest that the effect of selenium in protection from oxidative stress and
mitochondrial biogenesis is not only observed in trophoblast cells but also in first
trimester placental tissues.
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7.2
INTRODUCTION
Placental oxidative stress has been implicated in the pathogenesis of pre-
eclampsia (Burton et al., 2004) and this can lead to cell turnover and shedding of
placental debris into the maternal circulation. It has been established that mitochondria
are involved in placental oxidative stress through the generation of ROS which can
cause cell death through apoptosis or necrosis (Orrenius et al., 2007). Hypoxia and reoxygenation are the major factors which stimulates ROS production in placenta
(Burton, 2009).
A number of studies have looked at the function of placental mitochondria,
where researchers have used both whole placenta and specific cell populations
(Maloyan et al., 2012, Mandò et al., 2014). The work in this chapter investigates
mitochondrial respiratory function in the syncytiotrophoblast of first trimester
placental tissue that has been cultured as whole tissue explants rather than isolated
cells,
thus
preserving the relationship of the placental cell
types.
The
syncytiotrophoblast is the primary source of placental debris which is shed into the
maternal circulation and this debris are thought to have a key role in the
pathophysiology of pre-eclampsia (Abumaree et al., 2006). The expression of antioxidant systems, mitochondrial biogenesis and mitochondrial content has been
investigated in whole placental tissue to give an overview of general placental health.
Placental tissue from women with pre-eclampsia demonstrates higher levels of
ROS. Several studies have been conducted which suggest that placental tissues of preeclamptic women are in significant oxidative stress (Das et al., 2012, Burton et al.,
2011). Due to increased placental oxidative potential in pre-eclamptic mothers, there is
increased ROS generation which overwhelms cellular anti-oxidant defence system.
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Increase
ROS
production
mediates
apoptosis
in
placenta,
which
causes
syncytiotrophoblast necrotic shedding (Myatt et al., 2004).
In Chapter 4 and 5 of this thesis it was shown that selenium treatment rescued
the detrimental effects of oxidative stress and apoptosis in trophoblast -like cell lines
when endogenous stress was applied. However, it is yet to be determined if this might
occur in placental tissues. In this chapter we investigated the effects of short term
selenium supplementation on the expression key anti-oxidant proteins and
mitochondrial biogenesis markers. The effects on mitochondrial number and function
were also examined. These experiments were designed to validate our studies in
another model and show the beneficial effect of selenium treatment on villous
placental explants. This is an important consideration when developing potential
treatment regimens for complications of pregnancy such as pre-eclampsia but might
also inform in-vitro techniques for explant culture and led to improved experimental
conditions for conducting these studies.
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7.3
AIMS
The specific aims of this study were:
1
To determine the effect of selenium supplementation on the protein expression
of GPx and Thx-Red in placental tissues using western blotting.
2
To determine the effect of selenium supplementation on the mRNA expression
of GPx and Thx-Red in placental tissues using qPCR.
3
To measure the expression of mitochondrial biogenesis proteins selenoprotein
H, PGC-1α and NRF-1 cultured with or without selenium by western blotting.
4 To measure the mitochondrial content of villous placental explants cultured with
or without selenium using qPCR.
5
To measure mitochondrial respiration of the syncytiotrophoblast of first trimester
placental tissues cultured with or without selenium using an Oxygraph-2k
Oroboros high respirometry chamber.
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7.4
MATERIALS AND METHODS
7.4.1 Tissue collection and culture
This study was approved by the Human Research Ethics Committee, Metro
North Hospital and Health Service, Queensland Government, Australia and the
Auckland Regional Ethics Committee, New Zealand and all tissues were obtained
following written informed consent (Ethics approval number HREC/14/QPCH/249).
Third trimester placental tissue was obtained from 11 pregnancies (37.2–40.9 weeks of
gestation) following elective caesarean section. For term tissue, samples were taken
from six random locations on the maternal aspect. The placental tissue was collected
within 15 minutes of delivery and immediately transported to the laboratory. The
tissue was rinsed with PBS and 10–40mg wet weight of placental explants were
cultured at 37°C with 5% CO2 and 21% O2 in Dulbecco’s Modified Eagle Medium
(Invitrogen, New Zealand) containing 10% fetal bovine serum and 100U/L penicillin.
Matched explants of villous tissue were treated with Sodium Selenite (NaSe) at 100nM
or PBS vehicle control in culture for increasing times as detailed below.
First trimester placental tissue was obtained from 19 pregnancies (7.2–13
weeks of gestation) following elective surgical termination. Samples were randomly
selected; for first trimester tissue. The termination for pregnancy procedure destroys
macroscopic tissue architecture (that is, it all gets torn apart), so samples are
necessarily random. Tissue was collected within 10 minutes of delivery and
transported to the laboratory. The tissue was rinsed with PBS and immediately
transferred to Histidine-tryptophan-ketoglutarate (HTK) transplant solution (Essential
Pharmaceuticals LLC, USA) on ice and taken to the laboratory. 10–40mg wet weight
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of placental explants were cultured at 37°C with 5% CO2 and 21% O2 in Dulbecco’s
Modified Eagle Medium (Invitrogen, New Zealand) containing 10% fetal bovine
serum, 5ng/mL epidermal growth factor, 5μg/mL insulin, 10μg/mL transferrin, 400U/L
human chorionic gonadotropin, 100μg/mL streptomycin and 100U/L penicillin. For
both first trimester and term tissue the samples were then pooled. The tissue samples
were all from non-labouring women. The tissues were not karyotyped and this was not
something covered by our ethics. Matched explants of villous tissue were treated with
100nM NaSe or PBS vehicle control in culture for 4, 12, 14, 24, 48 or 96 hours.
The first trimester tissue experiments were conducted in collaboration with Dr
Olivia Holland and Prof Larry Chamley, University of Auckland.
7.4.2 Western Blotting
Western blotting was performed on placental tissues as described in section 2.7
and protein concentration was measured as detailed in section 7.4.3. The protein
extracts of placental tissues which were treated with 100nM NaSe at 4, 12, 24, 48 and
96 hours were probed with Sel H (Santa Cruz Biotechnology, USA), PGC-1α (Cell
Signaling Technology, Australia), NRF-1 (Cell Signaling Technology, Australia), and
β-actin (Cell Signaling Technology, Australia). The protein extracts of placental
tissues which were treated with 4 hours and 12 hours of 100nM NaSe were probed
with Glutathione Peroxidase 1 (Abcam, Australia) and Thioredoxin-Reductase 1
antibody (Cell Signaling Technology, Australia) at 1:1000 dilutions overnight at 4°C
shaker followed by consecutive washes with TBST. The membranes were incubated
with horseradish peroxidase-conjugated goat anti-rabbit secondary IgG antibody at a
dilution of 1:5000 (Santa Cruz Biotechnology, USA) or horseradish peroxidase-
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conjugated mouse anti-goat secondary IgG antibody at a dilution of 1:5000 (Santa
Cruz Biotechnology, USA) in 5% BSA and 0.1%Tween-20 for 1 hour at 37°C on a
shaker. The processing of the blots and densitometry analysis was completed as
detailed in methods section 2.7.
7.4.3 DNA, RNA and protein extractions
DNA, RNA and protein extractions of all placental explants were performed
using the All Prep DNA/RNA/Protein Mini Kit (Qiagen, Australia) according to the
manufacturer’s protocol. DNA and RNA concentration were measured on the 2000c
Nanodrop (Thermo Scientific). The protein concentrations were measured by Direct
Detect® Spectrometer (Merck Millipore, Australia).
7.4.4 cDNA synthesis
cDNA was synthesized from extracted RNA using QuantiTect Reverse
Transcription kit (Qiagen, Australia). Briefly, 1μg of purified RNA was combined with
7x genomic DNA Wipeout Buffer (Qiagen, Australia) with the final volume made up
to 14μL with RNase-free water. This mix was incubated for 2 minutes at 42°C and
immediately placed on ice. A reverse-transcription master mix was prepared for each
sample with 1μL of Quantiscript Reverse Transcriptase (Qiagen, Australia), 4μL of 5x
Quantiscript RT buffer (Qiagen, Australia) and RT Primer mix (Qiagen, Australia).
The RNA template mix was added in each tube containing reverse-transcription master
mix and was incubated at 42°C for 15 minutes to synthesized cDNA. The reaction mix
was further incubated for 3 minutes at 95°C to inactivate Quantiscript Reverse
Transcriptase, and was stored at -20°C until further use.
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7.4.5 Real time Polymerase Chain Reaction (RT-qPCR) for determining the
expression of anti-oxidant enzymes
Quantitative real-time PCR to determine the expression of the anti-oxidant
enzymes GPx1 and Thx-Red1 was conducted on RNA extracted from cells and tissue
using the QuantiFast SYBR Green PCR master mix on a Rotor Gene PCR Machine
(Qiagen, Australia). The final reaction volume of 12.5μL contained 200nM of reverse
and forward primers (Table 7.1), 6.25μL SYBR Green master mix (Qiagen, Australia),
2.25μL of RNase-free water and 2μL of cDNA of an optimised dilution (1/25 for
GPx1, Thx-Red1 and MTBA). The PCR profile consisted of a 95°C step for 5 minutes
followed by 40 cycles consisting of 30 seconds at 95°C, 45 seconds at 64°C and 30
seconds at 72°C. PCR amplification efficiency was tested in all assays by pooling
cDNA as a cross-run calibrator at a serial dilution of 1/100, 1/200, 1/400. Nontemplate controls (NTC) were also tested to determine any contamination. The CT
values were normalised to the MTBA CT values of the same cDNA sample. Fold
differences of each sample was calculated by 2−ΔΔCt method.
Table 7.1. Real time PCR primers
Genes
Forward Primer
Reverse Primer
GPx1
CCC GTG CAA CCA GTT TGG
TGA AGT TGG GCT CGA AC CC
Thx-Red1
GAC CAA AAA GCA GCT GGA CAG
GGA GGA TGC TTG CCC CA
MTBA
AGC GGG AAA TCG TGC GTG AC
AGG CAG CTC GTA GCT CTT CTC
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7.4.6 Real time Polymerase Chain Reaction (RT-qPCR) for determining
mitochondrial DNA copy number.
Real-time quantitative PCR (RT-qPCR) was performed on DNA extracted
from placental tissue supplemented with 100nM NaSe at 4, 12, 24, 48 and 96 hours, as
described for cells in section 2.5. 25ng/μL of total DNA was used in combination with
10μM of forward and reverse primers (Table 7.2). The threshold values (Ct) of
samples were determined using QuantiFast SYBR Green PCR master mix (Qiagen,
Australia) on a Rotor Gene PCR Machine (Qiagen, Australia). The quantity of four
mitochondrial genes (Table 7.2) was normalised to single-copy nuclear gene (Table
7.2) to give the ratio of the number of mitochondrial genomes per nuclear genome. An
internal assay reference gene (β-actin) was also included. Relative quantification
values are calculated using 2−ΔΔCT, where ΔΔCT compares each mitochondrial gene
with the nuclear gene and the final calculation expresses this as a ratio change based
on amplification (Schmittgen et al., 2008).
Table 7.2. Real time PCR primers
Genes
Target
Forward Primer
Reverse Primer
MTRT1
mtDNA
CAC CCA AGA ACA GGG TTT GT
TGG CCA TGG GTA TGT TGT TA
MTRT2
mtDNA
TCC TCC TAT CCC TCA ACC CC
CAC AAT CTG ATG TTT TGG TTA AAC
MTRT3
mtDNA
CAT CTG GTT CCT ACT TCA GGG
TGA GTG GTT AAT AGG GTG ATA GA
MTRT4
mtDNA
ATG GCC CAC CAT AAT TAC CC
CAT TTT GGT TCT CAG GGT TTG
MTBA
nDNA
AGC GGG AAA TCG TGC GTG AC
AGG CAG CTC GTA GCT CTT CTC
nDNA
GAG TTT CCT GGA CAAATG AG
CAT TGT TTC ATA TCT CTG GCG
MTAIB
mtDNA=mitochondrial DNA; nDNA=nuclear DNA.
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7.4.7 Measurement of mitochondrial respiration
Mitochondrial respiration was measured in an Oxygraph-2k chamber with
matched explants of villous placental tissues supplemented with or without 100nM
NaSe for 4, 12, 14, 24, 48 or 96 hours. Measurements were also made at time zero,
defined as the time tissues arrived at the laboratory. The tissues were permiabilized
with 50μg/mL saponin in 1mL BIOPS solution (2.77mM CaK2EGTA, 7.23mM
K2EGTA, 5.77mM Na2ATP, 6.56mM MgCl2⋅6H2O, 20mM taurine, 15mM Na2
phosphocreatine, 20mM imidazole, 0.5mM dithiothreitol, 50mM MES, pH 7.1) for 30
min at 4°C. Explants were then washed twice for 10 min at 4°C in MiRO5 respiration
medium (0.5mM EGTA, 3mM MgCl2·6H2O, 60mM K-lactobionate, 20mM Taurine,
10mM KH2PO4, 20mM HEPES, 110mM Sucrose, 1g/L BSA pH 7.1) and blot-dried
before measuring wet weight. 10–40mg wet weight of tissue was added to an
Oxygraph-2k chamber containing MiRO5 at 37°C and SUIT protocol was used to
analyse the function of different mitochondrial complexes. Pyruvate (5mM) and
malate (2mM) were added to determine C I-mediated LEAK respiration and then ADP
(1.25mM) was added to stimulate oxidative phosphorylation OXPHOS. To test the
integrity of outer mitochondrial membrane Cytochrome c (10M) was added. The
addition of Rotenone (1M) inhibited C I. Succinate (10M) was then used to
stimulate OXPHOS through C II, followed by titrations of FCCP (0.5M) to
investigate uncoupling of the mitochondria. The addition of Antimycin A (5M)
inhibited C III. N,N,N',N'-Tetramethyl-p-phenylenediamine dihydrochloride (TMPD;
0.5mM) and ascorbate (2mM) were added to determine OXPHOS through complex IV
(C IV).
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7.4.8 Statistical Analysis
All data are presented as mean ± S.D. with P<0.05 considered significant.
Statistical analysis was performed using the Graph Pad Prism version 6.0 statistical
package (GraphPad, USA). Kruskal-Wallis with Dunn's multiple comparisons was
used in Figure 7.7 and a two-tailed test was used in Figure 7.1, Figure 7.2, Figure 7.3,
Figure 7.4, Figure 7.5, Figure 7.6, Figure and Figure 7.8 to analyse the difference
between the treatments. Grubb's test for outliers was used as the basis for exclusion.
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7.5
RESULTS
7.5.1
Expression of GPx and Thx-Red anti-oxidant enzymes in first trimester
placental tissues
Western blot analysis for the anti-oxidant enzymes GPx 1 and Thx-Red 1 was
performed on placental tissues collected at time of delivery from 1st trimester
terminations. Tissues explants were treated with 100nM NaSe for 4 hours and 12
hours. A significant increase was observed in both GPx and Thx-Red expression when
tissues were supplemented with 100nM NaSe for 4 hours as compared to the controls
(Figure 7.1). A fold increase of 3.08 (P<0.05) for GPx and 2.15 (P<0.05) for Thx-Red
expression was observed. At 12 hours of selenium treatment, there did not appear to be
an increase in GPx and Thx-red expression.
Figure 7.1 GPx and Thx-Red expression in 1st trimester placental tissues treated with
100nM NaSe for 4 and 12 hours compared to controls. Images show
representative western blot bands. Plots show densitometry expression
levels of GPx and Thx-Red normalised to actin expression. Data represents
mean ± S.D. CNTL=control ;( *p<0.05), n=3.
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7.5.2 GPx and Thx-Red anti-oxidant enzymes in term placental tissues
Western blot analysis of GPx and Thx-Red was performed on term (37.2–40.9
weeks of gestation) placental tissues collected at time of delivery (elective caesarean
section). Tissues explants were treated with 100nM NaSe for 4 hours or vehicle
control. A significant increase was observed in GPx expression in the selenium treated
samples relative to the untreated control (fold 2.83; P<0.05). Thx-Red expression
significantly (P<0.05) decreased after 4 hours in culture and no significant difference
was observed between the selenium treated samples and controls (Figure 7.2).
Figure 7.2 GPx and Thx-Red expression in term placental tissues immediately after
collection (Time 0) and after treatment with 100nM NaSe for 4 hours
compared to controls. Images show representative western blot bands.
Plots show expression levels of GPx and Thx-Red normalised to actin
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expression. Sample 1,2,3 represents individual placentae. Data represents
mean ± S.D.; CNTL=control (*p<0.05), GPx-1 n=6, Thx-Red n=3.
7.5.3 mRNA expression of GPx and Thx-Red in first trimester placental tissues
Real-time PCR was conducted on placental tissues which were treated with
100nM NaSe for 4 hours and 12 hours to determine the gene expression for the antioxidant enzymes GPx and Thx-Red. Tissues treated for 4 hours with selenium showed
a significant increase in expression of GPx and Thx-Red versus control tissues (Figure
7.3). For GPx expression, there was a significant fold increase of 2.34 (P<0.05)
whereas in Thx-Red the fold increase of 3.24 (P<0.01) was observed. There were no
differences demonstrated at 12 hour selenium treatment. Similar experiments
conducted in term tissues showed considerable variability in results and are not
presented.
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Figure 7.3 GPx and Thx-Red mRNA expression in placental tissues treated with
100nM NaSe for 4 and 12 hours compared to controls. Data represents
mean ± S.D. (*p<0.05, **p<0.01) n=6.
7.5.4 Selenium upregulates mitochondrial biogenesis markers in first trimester
placental tissues
First trimester placental villous explants were supplemented with 100nM NaSe
for 4, 12, 24, 48 and 96 hours. It was found that selenium supplementation
significantly increases the expression of Sel H, PGC-1α and NRF-1 at early time
points (Figure 7.4, Figure 7.5 and Figure 7.6). At 4 hours and 12 hours of selenium
treatment, there was a significant increase in the expression of Sel H with fold increase
of 2.261 (P<0.05) (Figure 7.4). Expression of PGC-1α and NRF-1 after 4 hours of
selenium treatment was also increased by 3.55 (P<0.05) and 2.385 fold (P<0.05)
(Figure 7.5 and Figure 7.6). There were no significant differences of expression
observed on any mitochondrial biogenesis markers at 24, 48 and 96 hours of selenium
treatment.
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Selenium supplementation promotes mitochondrial function in placental tissues
Chapter 7
Figure 7.4 Sel H expression in placental tissues treated with 100nM NaSe for 4, 12,
24, 48 and 96 hours compared to controls. Images show representative
western blot bands. Plots show expression levels of Sel H normalised to
actin expression with untreated control samples then normalised to one to
give relative expression of Sel H in untreated control and selenium
supplemented samples. Data represents mean ± S.D. CNTL=control;
(*p<0.05), n=3.
148
Selenium supplementation promotes mitochondrial function in placental tissues
Chapter 7
Figure 7.5 PGC-1α expression in placental tissues treated with 100nM NaSe for 4, 12,
24, 48 and 96 hours compared to controls. Images show representative
western blot bands. Plots show expression levels of PGC-1 α normalised to
actin expression with untreated control samples then normalised to one to
give relative expression of PGC-1 α in untreated control and selenium
149
Selenium supplementation promotes mitochondrial function in placental tissues
Chapter 7
supplemented samples. Data represents mean ± S.D.; CNTL=control;
(*p<0.05), n=3.
Figure 7.6 NRF-1 expression in placental tissues treated with 100nM NaSe for 4, 12,
24, 48 and 96 hours compared to controls. Images show representative
western blot bands. Plots show expression levels of NRF-1 normalised to
actin expression with untreated control samples then normalised to one to
give relative expression of NRF-1 in untreated control and selenium
150
Selenium supplementation promotes mitochondrial function in placental tissues
Chapter 7
supplemented samples. Data represents mean ± S.D.; CNTL=control;
(*p<0.05), n=3.
7.5.5
Selenium may increase mitochondrial content in first trimester placental
tissues
Mitochondrial content was measured in first trimester villous tissues explants.
To observe the effect of selenium on mitochondrial number, placental tissues were
treated with 100nM NaSe for 4, 12, 24, 48 and 96 hours and real-time qPCR for
mitochondrial and nuclear genes was performed. Data are presented as mitochondrial
gene number normalised to nuclear gene number, to give a measure of mitochondrial
content. There were no significant fold differences observed in the 4 mitochondrial
genes investigated (MTRT1, MTRT2, MTRT3 and MTRT4) (Figure 7.7) as compared
to the control when normalised to the reference nuclear gene, β-actin. There was an
increasing trend observed in the mitochondrial content when tissues were
supplemented with selenium, however, it did not reach statistical level.
151
Selenium supplementation promotes mitochondrial function in placental tissues
Chapter 7
Figure 7.7 Mitochondrial content measured in placental tissues when supplemented
with selenium for 4, 12, 24, 48 and 96 hours. Values are presented as
means ± S.D. n=6, CNTL=control.
7.5.6
Selenium increases mitochondrial respiration in first trimester placental
tissues
(The following experiments were conducted in collaboration with Dr Olivia
Holland and Professor Larry Chamley, University of Auckland and presented here
with their permission).
Mitochondrial respiration was measured in first trimester villous tissues
explants. The tissues were treated with 100nM NaSe for 4, 12, 14, 24, 48 and 96 hours.
Explants were permiabilised to obtain access to syncytiotrophoblast mitochondria and
mitochondrial complexes-specific fluxes were determined.
Selenium
treatment
significantly
enhanced
oxidative
phosphorylation
(OXPHOS) through complex IV after four hours (P=0.001) of culture (Figure 7.8).
There was no significant increase observed in the other state-specific fluxes
investigated (LEAK, OXPHOS I and OXPHOS II) nor were significant increases
observed at longer time points of incubation at 24, 48 or 96 hours of treatment (data
not shown). OXPHOS IV represents the final step in the electron transfer system
(ETS), with electron donation from complex IV directly to O2 to form H2O, thus, the
selenium treated placental mitochondria had greater respiratory capacity. At the time
of writing, similar experiments in term tissue were displaying a similar trend but
statistical significance had not been demonstrated due to inter experiment variation.
152
Selenium supplementation promotes mitochondrial function in placental tissues
Chapter 7
Figure 7.8 Effect of Selenium treatment on oxidative phosphorylation through
complexes IV, II and I, and LEAK respiration in first trimester placental
tissues. Mitochondrial Respiration was measured after 4 or12 hours in
culture. At each time point the number of placentae investigated were 4
hours
n=11,
12
hours
n=5.OXP=
oxidative
phosphorylation;
CNTL=control ;( ***p<0.001).
153
Selenium supplementation promotes mitochondrial function in placental tissues
Chapter 7
7.6
DISCUSSION
There is a substantial literature in support of the hypothesis that placental over-
production of ROS and mitochondrial oxidative stress are central to the
pathophysiology of variety of diseases including cancer, vascular diseases and preeclampsia (Sosa et al., 2013, Madamanchi et al., 2005, Burton et al., 2004). Oxidative
stress is defined as an imbalance which is due to the excessive generation of reactive
oxygen species (ROS) through the partial reduction of molecular oxygen and this
needs to be negating by the actions of anti-oxidants. Our previous work in this area has
determined that selenium supplementation can up regulate anti-oxidant expression in
trophoblast cells providing protection from mitochondrial oxidative stress (Watson et
al., 2012, Khera et al., 2013). Furthermore, in Chapter 6 of this thesis it was reported
that selenium supplementation boosted mitochondrial respiration and mitochondrial
biogenesis in trophoblast cells. To extend our observations into another model, we
investigated the effect of selenium treatment (100nM NaSe) for 4, 12, 14, 24, 48 or 96
hours in villous placental tissue explants collected from 1st and 3rd trimester tissues.
As seen in trophoblast like-cell lines, selenium supplementation increased the
expression of GPx and Thx-Red in both term placental tissues and placental explants
from 1st trimester placental villi. The mRNA expression of the anti-oxidants GPx and
Thx-Red was also increased in placental tissues treated with selenium. However, there
appeared to be a time dependent effect as this increased expression was not observed
after 12 hours of treatment.
Treatment of villous placental tissues with 100nM NaSe for 4 or 12 hours also
led to a significant increase in the expression of Sel H, PGC-1α and NRF-1. This result
supports the observations made in Chapter 6 of this thesis which demonstrated that Sel
154
Selenium supplementation promotes mitochondrial function in placental tissues
Chapter 7
H, PGC-1α and NRF-1 is acutely sensitive to intracellular concentrations of selenium
and when present in higher concentrations Again there appeared to be a time
dependent aspect to this observation as significance was lost at longer time points
although a clear trend of overproduction can be seen. The increase in these biogenesis
factors was able to boost mitochondrial number when analysed with qPCR
amplification of mitochondrial genes verses nuclear genes. Although there was not a
statistically significant difference here, due to inter assay variation, there is a clear
trend with increased mitochondrial number seen in those tissues treated with NaSe.
Mitochondrial respiration was significantly enhanced through complex IV after
4 hours of 100nM NaSe treatment however this effect was not observed at longer time
intervals. There appeared to be a slight increase in respiration through complexes I and
II and overall LEAK respiration though this only appeared at the lower time points and
did not reach statistical significance. When tissue is collected post pregnancy
termination or term delivery there is a small window of time in which to process the
tissue and make experimental observations. This has been shown to be the case with
term tissue (Burton et al., 2014) and during our collaboration with Dr Olivia Holland
and Professor Larry Chamley. Dr Holland conducted the 1st trimester oxygraph
experiments and demonstrated that placental explants were most responsive at 4 and
12 hours post collection but lost functionality at longer time points (personal
communication). This seems to be the case with selenium responsiveness as well with
maximal effects seen at the shorter treatment time of 4 hours, occasionally at 12 hours
but significance was lost at 24 hours. As part of a broader study by Dr Olivia Holland
in our laboratory we have examined the viablity of tissue collected from first trimester
and from term placental tissue. Tissue processing should be completed with 30
155
Selenium supplementation promotes mitochondrial function in placental tissues
Chapter 7
minutes of collection and mitochondrial function shows evidence of decline after 8
hours in culture. These studies will be reported soon and are outside the scope of this
thesis. In cultured placental cells we used a standardised time point of 24 hours as this
was shown to adequately up-regulate protein expression and was a convenient
experimental time course to follow. We did not test shorter time points which may
have shown in immediacy of the effect of selenium supplementation.
An interesting observation from these experiments is the lack of expression of
Thx-Red in placental tissue within 4 hours of delivery. This decreased expression also
was unresponsive to selenium supplementation. There is unpublished data from this
laboratory showing that the thioredoxin system, both Thx and Thx-Red are downregulated during the last trimester of pregnancy and this may continue immediately
post-delivery. This warrants further investigation.
In summary, the work presented in this chapter strongly supports previous
observations that selenium supplementation in-vitro, increases production anti-oxidant
seleno-enzymes such as GPx and Thx-Red and the transcription factor Sel H. This led
to improved mitochondrial respiration and activated mitochondrial biogenesis through
induction of the mitochondrial biogenesis factors PGC1-α and NRF-1 in the placental
explants.
These results are important in context of placental oxidative stress, which is a
causative factor in increased trophoblast shedding from the placental can initiate the
maternal hyper immune response characteristic of pre-eclampsia (Smarason et al.,
1993, Knight et al., 1998). There is considerable published evidence that increased
levels of oxidative stress (Myatt, 2010) and reduced mitochondrial function
(Muralimanoharan et al., 2012) in the placenta are key features of this disorder.
156
Selenium supplementation promotes mitochondrial function in placental tissues
Chapter 7
Therefore, selenium may have therapeutic potential in pre-eclampsia. The incidence of
pre-eclampsia is negatively correlated with selenium status (Mariath et al., 2011) and
clinical trials have been conducted that show selenium supplementation can reduce
markers associated with the risk of pre-eclampsia (Rayman et al., 2014, Tara et al.,
2010). The data presented here demonstrate potential modes of action of selenium on
trophoblast mitochondria up-regulation of endogenous anti-oxidant systems and
increased mitochondrial content and show how selenium may be protective for preeclampsia, by improving the ability of trophoblast mitochondria to cope with oxidative
stress. Together these data suggest that selenium can enhance the anti-oxidant capacity
and improve mitochondrial respiration not only in trophoblast cells but also in first
trimester placental tissues.
157
CHAPTER 8
GENERAL DISCUSSION
General Discussion
Chapter 8
Pre-eclampsia is a life threating complication of pregnancy, which occurs after
20 weeks of gestation and complicates 3-8% of all pregnancies (Goel et al., 2013).
Despite modern advances in health care, pre-eclampsia still causes approximately
60,000 maternal deaths per year and imposes a significant burden on health care costs
in developed nations. There is no cure for pre-eclampsia and the only definitive
treatment that will resolve the symptoms of the illness is the removal of the placenta
from the uterus. Pre-eclampsia is described as a syndrome as it is a multisystem
disorder affecting both mother and baby (Uzan et al., 2011). The clinical features of
pre-eclampsia include acute hypertension, in addition to the appearance of proteinuria,
thrombocytopenia, chronic inflammation and on occasions fetal growth restriction.
The pathophysiology of pre-eclampsia is complex, where the primary cause is
abnormal placentation with defective invasion of spiral arteries by cytotrophoblast
cells. The spiral arteries fail to remodel which results in increased uterine arterial
resistance leading to hypoxia and periods of ischemia and reperfusion. This generates
rapid oscillations in the supply of oxygen which leads to oxidative stress (SánchezAranguren et al., 2014).Oxidative stress refers to an imbalance between Reactive
Oxygen Species (ROS) and anti-oxidants and occurs when the generation of free
radicals, including ROS, exceeds the anti-oxidant capacity of the cell (Buonocore et
al., 2010). There is substantial evidence which suggests that placental oxidative stress
plays a critical role in the pathogenesis of pre-eclampsia (Burton et al., 2011).
The expression and activity anti-oxidant enzymes in the placenta have been the
subjects of much research in recent years (Myatt et al., 2004). Protein-based antioxidants enzymes such as Glutathione Peroxidase (GPx), Thioredoxin-reductase (ThxRed), Superoxide Dismutase (SOD) and catalase along with non-protein anti-oxidants
159
General Discussion
Chapter 8
such as glutathione, maintain redox balance in all cells (Espinosa-Diez et al., 2015).
During normal pregnancy, these anti-oxidants are up-regulated to scavenge the
increased burden of ROS which if not countered would lead to oxidative stress.
Research conducted on anti-oxidant status in women suffering from pre-eclampsia has
found significant decrease in GPx, Thx-Red, SOD and catalase expression as
compared to the normal pregnancies (Vanderlelie et al., 2004, Wang et al., 1996,
Wiktor et al., 2000, Sharma et al., 2006). Furthermore, the expression of these proteins
in the syncytium appears to be diminished, making this cell layer more susceptible to
oxidative damage and shedding through apoptotic and necrotic processes. This would
lead to shedding of potentially immune stimulatory debris that could activate the
maternal endothelium and generate the symptoms of pre-eclampsia. Anti-oxidant
therapies in the form of Vitamin C and E have been trialled in the prevention of preeclampsia but have not proven to be effective (Roberts et al., 2010), hence the interest
in increasing the expression and activity of endogenous anti-oxidants in a safe manner
that will not harm mother or baby.
Glutathione Peroxidase and Thioredoxin- Reductase are seleno-proteins whose
activity can be controlled by the supply of selenium. Not only is selenium required in
the active site in the form of the key amino acid selenocysteine, but also a
selenoprotein is involved in the miss reading of the UGA codon, which leads to
selenocysteine insertion (Hondal, 2005).This response to selenium was confirmed in
Chapter 3 of this thesis where trophoblast-like cells showed an increase in upregulation of both GPx and Thx-Red activity when treated with sodium selenite or
selenomethionine (Figure 3.1 and 3.4). This provides an effective control mechanism
for protein expression, which could be applicable to humans, as selenium
160
General Discussion
Chapter 8
supplementation has been shown to be safe in humans and during pregnancy at the
very low doses required to increase GPx and Thx-Red expression.
This was further investigated in Chapter 4 of this thesis, when mitochondrial
oxidative stress in trophoblast-like cells was generated through Rotenone and
Antimycin treatment, which acts by blocking complexes I and III of electron transport
chain respectively. Rotenone and Antimycin were shown to increase the endogenous
production of ROS in a dose dependent manner and selenium supplementation
significantly reduced the mitochondrial oxidative stress generated in trophoblasts cells
as a result of this insult (Figure 4.7-4.10). These results showed that selenium
supplementation could combat oxidative stress by preventing ROS production and
increasing cell viability (Khera et al., 2013).
Selenium is an essential trace element, vital for normal growth and
reproduction in humans and animals and there is considerable evidence for the
importance of micro-nutrition and trace elements like selenium for a healthy
pregnancy (Mistry et al., 2012). Selenium supplementation is essential for the upregulation of anti-oxidant selenoproteins whose expression and activity can limit an
oxidative insult (Pieczyńska et al., 2015). Selenium has been shown to be essential for
reproductive health in several mammalian species (Maiorino et al., 1999) and selenium
deficiency is associated with the risk of pre-eclampsia (Rayman et al., 2003) and other
complications of pregnancy (Mariath et al., 2011).
It has now been established that selenium counters ROS accumulation and
mitochondrial oxidative stress which prevents biological oxidation and damage to
cells. A study undertaken by Watson et al. in 2012 demonstrated that supplementation
with selenium is effective in protecting trophoblast cells from oxidative stress when
161
General Discussion
Chapter 8
oxidants are applied exogenously to cells in culture. However, the data reported here in
Chapter 4 is more significant as the oxidative insult has been generated from within the
mitochondria and still selenium was able to offer protection. Oxidative stress is a
powerful inducer of apoptosis and necrosis in most cell types and leads to the intrinsic
activation of cell death pathways (Payne et al., 1995). Depending upon the stimulation,
apoptosis is induced by two pathways: the mitochondrial or intrinsic pathway, initiated
by free radical production; or the extrinsic pathway, induced by TNF receptors.
Apoptosis rates were found to be significantly elevated in cytotrophoblast from
preeclamptic pregnancies when both the apoptotic activation pathways were observed
(Crocker et al., 2003). The work undertaken in Chapter 5 of this thesis showed that
induction of apoptosis was achieved through the use of mitochondrial inhibitors of the
electron transport chain and that Rotenone caused oxidative stress and induced
apoptosis in trophoblast cells. Apoptosis was measured by Bcl-2 protein, which is an
anti-apoptotic protein and studies have found that there was lower expression of Bcl-2
in pre-eclamptic pregnancies (Rani et al., 2007). In our experimental models, Bcl-2
expression was significantly decreased in trophoblast-like cells post Rotenone
treatment indicating that mitochondrial oxidative stress was causing the intrinsic
activation of apoptosis and cell death (Figure 5.1). To further confirm apoptosis can be
induced by Rotenone, apoptosis was also measured by Annexin V staining followed by
flow cytometry (Figure 5.3). Annexin V stain binds to phosphotidyl-serine and can be
used as an early indicator of apoptosis (Van Engeland et al., 1998). Trophoblast cells
exposed to Rotenone treatment significantly lost their membrane integrity upon
staining with Annexin V which confirmed the link between oxidative stress and
apoptosis. Apoptosis is found in placentas throughout the gestation period however,
162
General Discussion
Chapter 8
the rate of apoptosis is enhanced in pre-eclamptic placentas (Allaire et al., 2000).
Excess apoptosis in the placentae of pre-eclamptic women is associated with increased
trophoblast turnover and increased shedding of placental debris into the maternal
circulation (Sharp et al., 2010).
The protection from apoptosis through selenium supplementation with Sodium
Selenite (NaSe) was then examined. With selenium being essential for the expression
of anti-oxidants such as; GPx and Thx-Red (Rayman, 2000), the rationale behind the
selenium supplementation was to reduce ROS production in trophoblast cells, thereby
decreasing the degree of apoptosis. When supplementation with NaSe was introduced
in combination with Rotenone and it was observed that Bcl-2 expression was
significantly up-regulated in trophoblast-like cells (Figure 5.1 and 5.2). There was also
a shift observed in the population of cells supplemented with NaSe upon Annexin V
staining indicating a reduction in the amount of apoptosis (Figure 5.3). These results
are significant, as following treatment with NaSe, trophoblast cells have been able to
handle the oxidative insult caused by Rotenone and limit the progression of apoptosis
(Murphy, 2009). Similar observations were shown in other studies where it was
reported that selenium protected human keratinocytes from undergoing apoptosis
(Rafferty et al., 2003).
In summary, mitochondria are associated with ROS generation that causes
increased mitochondrial oxidative stress in cells, resulting in the activation of apoptotic
cell death pathways. The work in this thesis has shown that selenium supplementation
is a useful means to boost the anti-oxidant capacity and thereby increase mitochondrial
ability to combat oxidative stress (Khera et al., 2013). Excessive ROS production leads
to dysfunctional mitochondria which induce apoptosis in cells and can lead to tissue
163
General Discussion
Chapter 8
necrosis (Fleury et al., 2002). The experiments presented in this thesis strongly
indicate that selenium maybe important in preserving tissue integrity in diseases which
have oxidative stress as part of their pathophysiology, such as complications of
pregnancy such as pre-eclampsia.
Perhaps the most important findings during these studies are presented in
Chapter 6 of this thesis which clearly showed that selenium supplementation could
influence mitochondrial respiratory function and mitochondrial biogenesis in
trophoblast cells. Mitochondrial biogenesis is the process of growth of new
mitochondria in response to cellular stress. Mitochondrial proteins are encoded by both
nuclear and mitochondrial genomes which controls the transcription and translation of
the mitochondrial proteins (Jornayvaz et al., 2010). PGC-1α (Peroxisome proliferatoractivated receptor)–γ-coactivator-1α and NRFs (nuclear respiratory factors) 1 and 2 are
the major regulators of mitochondrial biogenesis (Bergeron et al., 2001) and selenium
has been shown to activate the phosphorylation of phosphatidylinositol 3-kinase
(PI3K)-AKT, a crucial regulator of PGC-1α (Yoon et al., 2002).
In Chapter 6 of this thesis, it was shown that selenium enhanced the
mitochondrial respiration in trophoblast-like cells (Figure 6.1). Selenium also
stimulated mitochondrial biogenesis through Selenoprotein H, which further activated
PGC-1α and NRF-1(Figure 6.3 and 6.4). These results demonstrate that selenium
supplementation
significantly
increases
mitochondrial
biogenesis
markers,
mitochondrial content and improves the mitochondrial functional performance (Khera
et al., 2015). To validate our observations into another model the effect of selenium
treatment was investigated in placental villous tissues. As shown in Chapter 7 of this
thesis, tissue explants confirmed that selenium increased mitochondrial respiration
164
General Discussion
Chapter 8
(Figure 7.8) and mitochondrial biogenesis through Selenoprotein H and transcription
factors; PGC-1α and NRF-1 in first trimester and term placental tissues (Figure 7.47.6). Furthermore, protein expression and mRNA expression of GPx and Thx-Red
were also enhanced in placental tissues post selenium supplementation (Figure 7.1,
7.3). The importance of these observations is linked to oxidative stress in placenta
which
causes
complicated
pregnancy
like
pre-eclampsia
where
selenium
supplementation might provide a beneficial therapy.
The National Health and Medical Research Council of Australia has published
that the recommended dietary allowances (RDA) of selenium for pregnant women is
60μg/day and an additional 10μg/day for breastfeeding infants. Selenium deficiency
has been associated with an increased incidence in pre-eclampsia (Rayman et al.,
2014). Interestingly, studies on rodents have also confirmed that when the pregnant
rats were given selenium depleted diets symptoms of pre-eclampsia developed
(Vanderlelie et al., 2004). An excellent meta-analysis by Mariath in 2011, brought
together data from 33 studies which present conclusive evidence that selenium
depletion was associated with serious complications of pregnancy including
spontaneous miscarriage, pre-term delivery and hypertensive disorders of pregnancy
such as pre-eclampsia. We have now explained these observations by demonstrating
the role of selenium in synthesising anti-oxidants such as GPx and Thx-Red and the
importance of these anti-oxidants in human placenta in combating the placental
oxidative stress (Vanderlelie et al., 2005, Mistry et al., 2008). Additionally, we have
also shown the dual benefit of selenium supplementation on improving mitochondrial
function and biogenesis through the recently discovered selenoprotein-H. These
165
General Discussion
Chapter 8
studies will underpin the development of potential therapies for treating these
complications of pregnancy.
Recently, several small clinical trials have been conducted to explore the
benefit of selenium supplementation in treating women at risk of developing preeclampsia. In 2010, Tara et al. reported treating 166 prim gravid women with 100μg
selenium per day from the 1st trimester to term. There was no incidence of preeclampsia in the test group and 3 in the control group (Tara et al., 2010). In a follow
up study 230 women were treated with either placebo of 60μg per day from 12 -14
weeks until term and the effects on some of the biomarkers of pre-eclampsia were
tested (Rayman et al., 2015). This study found a negative correlation between
selenium status and plasma levels of soluble sFLT-1, a marker of pre-eclampsia.
Whilst these studies were not powered to show definitive changes in the incidence of
pre-eclampsia they do provide evidence that selenium supplementation at 60-100μg
per day is safe and sufficient to maximise the expression of key selenoprotein enzyme
systems.
Many pregnant women take a multi-nutrient supplement, and the majority of
these contain 60-100μg of selenium. In a study from our laboratory (Vanderlelie et al.,
2014), we demonstrated that there was a lower incidence of pre-eclampsia in women
taking a multi-nutrient supplement when compared to women not taking a supplement
and this effect was amplified in women with a higher BMI. In this cohort of
approximately 1000 pregnancies the incidence of pre-eclampsia in the control group
was 2.7% whilst in the group of women taking a broad spectrum multi nutrient
supplement it was 1.2%. When BMI was taken into account, women with a BMI of
>30 and not taking a supplement the incidence of pre-eclampsia was 4.2 % but fell to
166
General Discussion
Chapter 8
1.7% in large women taking a supplement (Vanderlelie et al., 2014). Several other
reports have suggested that broad-spectrum multi-nutrient supplementation may be of
benefit in lowering the incidence of pre-eclampsia (Bodnar et al, 2006, Catov et al,
2009). It has been shown that obesity is associated with oxidative stress and the
presence of adipose tissues causes generation of ROS (Marseglia et al., 2014). The
mitochondrial and peroximal oxidation of fatty acids produces ROS in oxidation
reactions (Fernández-Sánchez et al., 2011). The increase in metabolic activity of
placental mitochondria results in enhanced production of ROS, which becomes
amplified with complicated pregnancies such as pre-eclampsia, gestational diabetes
and maternal obesity (Maloyan et al., 2012). Deficiency of selenium during pregnancy
leads to other complications of pregnancy other than pre-eclampsia such as improper
foetal development, low birth weight, still birth and pre-term birth (Darnton et al.,
2015). Whilst it is not possible to say that any beneficial effect of taking a multinutrient supplement is directly due to selenium, further research is warranted into the
beneficial effects of these products in preventing the incidence and severity of
hypertensive disorders of pregnancy.
In a related study, our research group established a global map of selenium
status using published plasma selenium levels and compared this to the incidence of
pre-eclampsia. In over 45 countries, encompassing several million pregnancies, there
was a significant negative correlation between plasma selenium status and the reported
incidence of pre-eclampsia in the population. Countries with very low selenium status
of < 50μg/L had higher incidences of pre-eclampsia. New Zealand and Finland are of
interest as those countries have actively intervened to increase the selenium intake of
their population through a campaign of using selenium based fertilisers and
167
General Discussion
Chapter 8
supplementing animal feeds with selenium. Over a period from the 1980’s to the
1990’s the selenium status of both countries almost doubled from 50-60μg/l to
>100μg/L in that time the reported incidence of pre-eclampsia fell from 3.85% to
1.97% in Finland and from 6.65% to 1.94% in New Zealand (Vanderlelie et al., 2011).
This is very strong evidence that selenium status has an effect on the incidence of this
important and costly complication of pregnancy.
In these studies, we have used immortalised trophoblast cell lines in place of
primary cells. Transformed cell lines give a pure cell population which is valuable as it
provides a consistent sample and reproducible results. They are also very cost-effective
and omit the ethical concerns associated with the use of animal and human tissue.
Primary cell cultures isolated from source tissues provide an alternative to
immortalised cell lines as these cells retain phenotypic characteristics of their tissue of
origin whereas continuous passages of cell lines can cause genotypic and phenotypic
variation (Kaur et al., 2012). However, primary cells are isolated in limited numbers,
have limited life span and finite cell division and also require a written consent and
ethical approval which is difficult and time consuming and access to the appropriate
source tissues can be difficult.
In conclusion the data obtained in this thesis clearly indicates that placental
oxidative stress causes increased ROS production, which can compromise
mitochondrial function and lead to placental apoptosis. Increased apoptosis and
necrosis results in increased production of placental cell debris, which when released
into the maternal circulation activate the maternal endothelium and leads to the
symptoms of pre-eclampsia. Being able to safely control the oxidative balance in
mitochondria may provide a potential therapy for these hypertensive diseases of
168
General Discussion
Chapter 8
pregnancy. This can be done through selenium supplementation, which not only
increases the expression and activity of key anti-oxidant systems but increase
mitochondrial biogenesis and hence cellular respiratory capacity. This suggests that
selenium supplementation may provide a practical approach to the treatment of women
who are at risk of developing pre-eclampsia and this thesis provides the experimental
justification for larger randomised control trials.
169
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