The Role Of Oxidative Stress Following Antenatal Synthetic Glucocorticoid Therapy

by

Susmita Sarkar

A thesis submitted in conformity with the requirements for the degree of Master of Science Department of Physiology University of Toronto

© Copyright by Susmita Sarkar (2018) The Role Of Oxidative Stress Following Antenatal Synthetic Glucocorticoid Therapy

Susmita Sarkar

Master of Science

Department of Physiology

University of Toronto

2018

Abstract

We investigated the role of oxidative stress following sGC treatment in a guinea pig model of gestation through measures including: expression of antioxidant enzyme genes, protein carbonylation, and the glutathione ratio in the hippocampus, placenta, and liver of female and male fetuses treated with two courses of betamethasone in utero. Results indicated no significant differences in hippocampal or placental gene expression. Peroxiredoxin-6 expression was significantly downregulated in the male liver following sGC, but there were no differences in the female liver. There were no differences in protein carbonylation or glutathione ratio in the fetal placenta or liver. To investigate long-term outcomes of sGC exposure in utero, we used gene set enrichment analysis of post-natal day 40 female hippocampi, and found significant downregulation of a gene set for oxidative phosphorylation. These finding suggest that while the fetus is acutely protected from oxidative stress following sGC, there might be long-term programming of mitochondrial functions.

ii Acknowledgments

These past two years have collectively been the most fulfilling experience of my education thus far. I would like to thank the following people for their significant role in shaping my time in graduate school.

First and foremost, I would like to thank my research supervisor, Dr. Stephen Matthews for this opportunity. His support, guidance, and patience were all instrumental to my growth as a scientist. He welcomed my ideas, encouraged my independence, and challenged me to think critically. He taught me to never be afraid of the unknown, inspired me to not be discouraged by unexpected results, and educated me on proper scientific rhetoric. For all of this and more, I thank you.

Secondly, I would like to thank the members of my research supervisory committee, Dr. Beverly Orser and Dr. Patrick McGowan for their expert feedback. They both were instrumental to the development and progression of my project. Additionally, I would like to thank the members of my defense committee, Dr. Martin Post, Dr. Robert Levitan, and Dr. Andrea Jurisicova for their thoughtful questions of my research.

Third, I would like to thank Alice Kostaki, the lab manager for the Matthews Lab. Her vast knowledge and experience was an invaluable resource; I could always rely on Alice to help me brainstorm, troubleshoot, and answer questions that I had asked her ten times over. Her unique sense of humor and unmistakable laugh always lifted the mood and made for some unforgettable lunchtime conversations. And of course, thank you for all of the candy.

Fourth, I would like to thank the members of the Matthews lab with whom I shared my time in the lab with. Alex Mouratidis introduced me to the lab and performed the animal work for this study; I cherish his mentorship, and more importantly, his friendship. I was fortunate enough to be desk mates with Liz Eng; I could always turn to her for her encouragement and advice, be that for science, fitness, or skincare. Andrada Naghi and I began our graduate studies together but I ended up learning so much from her; she taught me to care about what really matters, to appreciate the outdoors, and most importantly, how to be myself. I would also like to thank Tam Lye, Dr. Hiro Hamada, Dr. Guinever Imperio, Dr. Aya Sasaki, Abigail Lee, and Bona Kim for their feedback, support, and friendship during my time at the lab.

Fifth, I would like to thank recent alumni of the Matthews lab: Dr. Vasilis Moisiadis, Dr. Andrea Costantinof, Mohsen Javam, and Maria Sqapi for their help and friendship. Sixth, I would like to thank the members of GASP council of 2016/2017 and 2017/2018. While there are too many of you to name, some of my most cherished memories of graduate school are from my time on GASP, and I know I have made friendships that will last for a lifetime. Seventh, I would like to thank Anna Roy and Anita Yen. Thank you for picking me back up when I was down; your friendship means the world to me. Eighth, I would like to thank the Department of Physiology, members of administration, and the Faculty of Medicine at UofT. My time in graduate school was facilitated by how well organized the department and program was.

iii Finally, I would like to thank my parents, Chandan and Mithu Sarkar. As a child, you instilled in me the value of learning, and made such great sacrifices to provide me with opportunities to further my education. This past year was the most difficult year of my life and without your support, trust, and continuous encouragement, this accomplishment would not have been possible. When I could not walk, you carried me. When I could not eat, you fed me. And when I could not keep going, you made me. This thesis is dedicated to you and I hope to continue to make you proud.

iv Table of Contents

ACKNOWLEDGMENTS ...... III TABLE OF CONTENTS ...... V

1.0.0 INTRODUCTION 1.1 CLINICAL SIGNIFICANCE OF SGC TREATMENT IN FETUS ...... 1

1.1.1 MATERNAL CORTISOL PHYSIOLOGY ...... 1 1.1.2 SYNTHETIC GLUCOCORTICOIDS (SGC) : MECHANISMS, USES, AND EFFICACY ...... 4 1.2.0 OXIDATIVE DAMAGE – CELLULAR PERSPECTIVE ...... 6

1.2.1 ROS – PHYSIOLOGICAL ROLE ...... 6 1.2.2 ANTIOXIDANT ENZYMES ...... 7 1.2.3 OXIDATIVE DAMAGE (MARKERS) ...... 8 1.2.4 OXIDATIVE STRESS PROGRESSION...... 9 1.3.0 CLINICAL SIGNIFICANCE OF OXIDATIVE DAMAGE IN UTERO ...... 10

1.3.1 NORMAL PREGNANCY - BASELINE ROS ...... 10 1.3.2 ROS LEADING TO PREGNANCY COMPLICATIONS...... 11 1.3.3 LONG-TERM OUTCOMES LATER IN THE LIFE OF THE CHILD ...... 13 1.4.0 ESTABLISHING MECHANISM BETWEEN GC AND OXIDATIVE STRESS – IN VITRO ...... 16 1.5.0 EFFECTS OF GC ON OXIDATIVE STRESS, IN VIVO ...... 18 1.6.0 EFFECTS OF PRENATAL GC ON OXIDATIVE STRESS IN FETUS/ LATER OUTCOMES IN LIFE ...... 20 FIGURE 1.1 CELLULAR OXIDATIVE STRESS ...... 23

2.0.0 OBJECTIVES, RATIONALE, AND HYPOTHESIS 2.1 OBJECTIVE: ...... 24 2.2 RATIONALE: ...... 24 2.2.1 BRAIN – HIPPOCAMPUS ...... 24 2.2.2 PLACENTA ...... 25 2.2.3 FETAL LIVER ...... 26 2.3 HYPOTHESIS ...... 27

3.0.0 METHODS 3.1 ANIMAL PROTOCOL ...... 28 3.2 QUANTITATIVE REAL-TIME PCR (QRT-PCR) ...... 29 3.2.1 PRIMER DESIGN, STANDARDS, EFFICIENCY ...... 29 3.2.2 TISSUE PREPARATION, RNA EXTRACTION AND CDNA CONVERSION ...... 30 3.2.3 PCR, HOUSEKEEPING GENES, LIST OF GENES ...... 31 3.3 PROTEIN CARBONYLATION ...... 32 3.3.1 PROTEIN EXTRACTION, STORAGE TIMES/CONDITIONS ...... 32 3.3.2 BRADFORD...... 32

v 3.3.3 PROTEIN CARBONYLATION...... 32 3.3.4 BCA PROTEIN ASSAY...... 34 3.3.5 CALCULATIONS ...... 34 3.4 GLUTATHIONE ASSAY ...... 35 3.4.1 TISSUE PREP FOR GLUTATHIONE ...... 35 3.4.2 GLUTATHIONE ASSAY ...... 35 3.4.3 CALCULATIONS ...... 36 3.5 POSITIVE CONTROL ...... 36 3.6 GENE SET ENRICHMENT ANALYSIS ...... 37 3.7 STATISTICAL ANALYSES ...... 37 TABLE 3.1 PRIMER SEQUENCES...... 38 TABLE 3.2 STABILITY OF HOUSEKEEPING GENES ...... 39

4.0.0 RESULTS 4.1 FETAL HIPPOCAMPUS ...... 40 4.2 FETAL PLACENTA ...... 40 4.3 FETAL LIVER ...... 41 4.4 MATERNAL HIPPOCAMPUS...... 42 4.5 COMPARATIVE GENE EXPRESSION ...... 42 4.6. ASTRCYTE EXPOSURE TO H2O2 ...... 42 APPENDIX : GENE SET ENRICHMENT ANALYSIS OF PND40 FEMALE HIPPOCAMPUS ...... 43 FIGURE 4.1.0 - FETAL HIPPOCAMPUS GENE EXPRESSION ...... 44 FIGURE 4.2.1 - FETAL PLACENTA GENE EXPRESSION ...... 46 FIGURE 4.2.2 - FETAL PLACENTA PROTEIN CARBONYLATION ...... 48 FIGURE 4.2.3 - PLACENTA GLUTATHIONE (TOTAL) ...... 48 FIGURE 4.3.1 - FETAL LIVER GENE EXPRESSION ...... 49 FIGURE 4.3.2 - FETAL LIVER PROTEIN CARBONYLATION ...... 51 FIGURE 4.3.3 A - FETAL LIVER GLUTATHIONE (TOTAL) ...... 52 FIGURE 4.3.3 B - FETAL LIVER GLUTATHIONE (RATIO) ...... 52 FIGURE 4.4.0 - MATERNAL HIPPOCAMPAL GENE EXPRESSION ...... 53 FIGURE 4.5.0 - COMPARATIVE GENE EXPRESSION ...... 54 FIGURE 4.6.0 - ASTROCYTE GENE EXPRESSION ...... 55 APPENDIX 1: GENE SET ENRICHMENT ANALYSIS IN FEMALE PND40 HIPPOCAMPUS ...... 56

5.0.0 DISCUSSION 5.1 HIPPOCAMPUS, PLACENTA – GENE EXPRESSION ...... 57 5.2 LIVER – GENE EXPRESSION ...... 58 5.3 PLACENTA, LIVER- PROTEIN CARBONYLATION, GLUTATHIONE RATIO ...... 59

vi 5.4 MATERNAL HIPPOCAMPUS – GENE EXPRESSION ...... 60 5.5 COMPARATIVE GENE EXPRESSION IN TISSUES ...... 60

5.6 ASTROCYTE EXPOSURE TO H2O2 ...... 62 5.7 PHYSIOLOGICAL LEVELS OF ROS ...... 62 5.8 TIMING, MODE OF DELIVERY ...... 63 5.9 NUTRITION ...... 64 5.10 NO ACUTE EFFECT OF SGC IN FETUS ...... 65 5.11 LONG-TERM OUTCOMES : PND40 FEMALE HIPPOCAMPUS - GSEA ...... 66 5.12 LIMITATIONS ...... 68 5.13 FUTURE DIRECTIONS ...... 69 5.14 SIGNIFICANCE AND CONCLUSIONS ...... 71

6.0.0 REFERENCES ...... 73

vii

1. Introduction 1.1 Clinical significance of sGC treatment in fetus

1.1.1 Maternal Cortisol Physiology

Cortisol is normally produced as a result of activation of the hypothalamus-pituitary- adrenal (HPA) axis. In response to stress, the hypothalamus secretes corticotropin-releasing hormone (CRH) and arginine-vasopressin (AVP). This signals to the anterior pituitary to release adrenocorticotropic hormone (ACTH). In response to ACTH, the adrenal cortex produces cortisol (Kivlighan et al., 2008). Cortisol functions to increase blood sugar through increasing gluconeogenesis in the liver. This cortisol negatively feeds back onto the HPA axis which then suppresses further hypothalamic secretion of CRH and pituitary release of ACTH.

During gestation, the placenta becomes an important source of cortisol through increased

CRH production. In humans and primates, during gestational weeks 8-10 and onwards, placental production of CRH begins to increase, which raises maternal ACTH and cortisol production

(Mastorakos & Ilias, 2003). In the placenta, cortisol does not have the same negative feedback function as the HPA axis, but rather has a feed-forward effect to maintain and increase maternal and fetal cortisol levels through stimulation of placental CRH (Davis & Sandman, 2010).

Bioactive (free) cortisol levels in the maternal circulation remain relatively constant until gestational week 25 (Kivlighan et al., 2008). This control is due to the presence of CRH binding protein (CRHbp) in maternal plasma and fetal amniotic fluid. CRHbp has high affinity for CRH and reduces CRH bioavailability. During the third trimester (around gestational week 25), the

1 concentration of CRHbp falls to one third of earlier pregnancy levels resulting in increased ‘free’

CRH. This unbound CRH stimulates maternal ACTH secretion from the anterior pituitary. As noted earlier, ACTH from the pituitary increases adrenal secretion of cortisol. The fetal pituitary becomes active in mid-gestation, accounting for two thirds of fetal stress responses. Once active, fetal hypothalamic secretion of CRH leads to fetal pituitary ACTH secretion. The fetal adrenal transitional zone synthesizes cortisol de novo from gestational week 28 onwards (Mastorakos &

Illias, 2003).

During pregnancy, there is compensatory hypertrophy of the maternal anterior pituitary and adrenal gland to accommodate this increase in hormone production. During pregnancy, the half-life of cortisol is doubled through the binding of corticosteroid binding globulin (CBG) in the maternal liver, thus further sustaining elevated cortisol levels (Mastorakos & Ilias, 2003).

Throughout most of gestation, the fetus is largely protected from this rise in maternal cortisol by placental barrier functions in the trophoblast layer. Placental p-glycoprotein (P-gp) effluxes cortisol out of the placenta back into the maternal circulation in an ATP-dependent manner (Moisiadis & Matthews, 2014). Placental 11beta-hydroxysteroid dehydrogenase (HSD)-

2 inactivates cortisol into cortisone. In humans, these two barriers create a gradient of cortisol across the placenta between the mother and the fetus (Moisiadis & Matthews, 2014). During late gestation, placental 11b-HSD2 expression decreases and maternal cortisol production rises between 2-4 fold (Murphy et al., 2006; Mastorakos et Ilias, 2003). These two factors allow for increased transfer of maternal cortisol to the fetal circulation, which is known as the late- gestational maternal cortisol surge (Davis & Sandman, 2010).

2

The maternal cortisol surge is a key developmental trigger for the maturation of fetal organs, particularly of the fetal lungs, liver, gut, kidneys, skeletal muscle and brain (Fowden et al., 2016). This prepares the fetus for newborn life, priming the fetus for pulmonary gas exchange, hepatic gluconeogenesis, and thermogenesis. In cases of preterm birth (<34 weeks of gestation), the fetus is not exposed to this developmental trigger. Consequently, respiratory distress syndrome (RDS) is the leading cause of mortality in preterm infants (Moisiadis &

Matthews, 2014).

Synthetic glucocorticoids (sGC), dexamethasone and betamethasone, are used in cases of expected preterm birth to mimic the cortisol surge and to mature the fetal lungs prior to birth.

Currently, the recommended single-course of betamethasone is two doses of 12 mg, intramuscularly, 24 hours apart and dexamethasone is recommended at a single course of four doses of 6 mg, intramuscularly, 12 hours apart (Ballard & Ballard, 1995). Betamethasone and dexamethasone readily cross the placenta as they are not metabolized by 11b-HSD2, and can effectively reach fetal glucocorticoid-sensitive organs (Moisiadis & Matthews, 2014). Both betamethasone and dexamethasone are substrates of P-gp which has a much greater effect on sGC transfer than cortisol (Moisiadis & Matthews, 2014). Clinical administration of sGC is given in large doses to saturate the transporters, causing maximal sGC transfer across the placenta (Moisiadis & Matthews, 2014).

3 1.1.2 Synthetic Glucocorticoids (sGC) : Mechanisms, uses, and efficacy

Glucocorticoid receptors are expressed in the following human embryonic tissues by gestational weeks 8-10: liver, skeletal muscle, brain, heart (Pujols et al., 2002), and in the lungs by week 12 (Drake & Seckl, 2011). As previously mentioned, cortisol is an important developmental trigger; thus, accurate timing of the cortisol surge is required for appropriate development. However, when glucocorticoids are administered earlier in gestation as a therapeutic intervention in cases of preterm birth, this has been associated with several adverse long-term outcomes such as neurobehavioral problems, metabolic dysfunction, and increased cardiovascular risks.

Many studies have shown antenatal synthetic glucocorticoids to have a long-term effect on behaviour in children (Davis & Sandman, 2010). Animal and human studies have shown a clear association between prenatal administration of sGC and later onset of hyperactivity and attention dysregulation, symptomatic of attention deficit hyperactive disorder (ADHD).

Pregnant guinea pigs treated with multiple courses of betamethasone had female offspring which exhibited increased locomotor activity in an open field test (Owens & Matthews, 2007).

Children treated with one course of low dose sGC in utero were more likely to exhibit symptoms of ADHD at 8 yrs and similar behaviour 16 yrs of age (not statistically significant) (Khalife et al., 2013). These findings have been attributed to hippocampal sensitivity to glucocorticoids

(Conrad, 2008). The fetal brain, particularly the forebrain and limbic structures, express high levels of glucocorticoid and mineralocorticoid receptors late in gestation when sGC are administered (Conrad, 2008; Matthews, 1998). The hippocampus has been shown to exhibit

4 global epigenetic changes following sGC treatment, leading to altered transcription of glucocorticoid responsive genes (Crudo et al., 2013).

Adverse metabolic outcomes following increased prenatal exposure to glucocorticoids include increased risk of altered muscle and adipocyte development, fatty liver disease, as well as dysregulated glucose and insulin homeostasis (Drake & Seckl., 2011). Rodents that were prenatally treated with dexamethasone and then fed a high-fat diet did not exhibit increased obesity or insulin resistance, but did show increased liver-triglyceride content (Drake et al.,

2010). However, there were few changes in transcript levels of genes involved in lipid metabolism in the liver and adipose tissue (Drake et al., 2010). Following prenatal sGC treatment in guinea pigs, male fetal liver showed significant reductions in global methylation at

GD52, prior to the natural cortisol surge (term ~ GD70) (Crudo et al., 2012). However, if sGC- prenatally treated offspring were delivered after the natural cortisol surge at GD65, male fetal liver showed increased global methylation patterns compared to controls (Crudo et al., 2012).

These findings suggest that sGC might be altering epigenetic patterns in metabolic tissues which are programming metabolic dysregulation later in life following a second stressor, for example a high fat diet.

Prenatal sGC has been associated with hypertension later in life of the offspring. In lambs following prenatal dexamethasone exposure, blood pressure control is dysregulated through changes in baroreflexes soon after birth, but at this time, measures of cardiac function are not altered (Dodic et al., 2002). However, by 4 months post-natal life, hypertension develops suggesting that symptomatic cardiac dysregulation occurs later in life (Dodic et al., 2002). One

5 study followed humans treated with prenatal sGC treatment until 30 years of age. While they did not find significant differences in cardiovascular risk between sGC treated and non-sGC treated subjects, they did however observe differences in markers of central insulin resistance in the treated group (Dalziel et al., 2005).

1.2.0 Oxidative Damage – cellular perspective

1.2.1 ROS – Physiological Role

Fetal growth is linear until 37 weeks, when it plateaus (Kivlighan et al., 2008). During this time, the cell cycle is going through rapid turnover. Reactive oxygen species (ROS) are

•- produced as a by-product of normal cellular reactions, including superoxide free radical (O2 ),

- hydroxyl free radical (OH ), and hydrogen peroxide (H2O2) (Sauer et al., 2001). These ROS are produced through: the mitochondrial respiratory chain, (NADPH)-oxidase complexes, phagocytotic cells where ROS act as part of defence system, other cells where ROS act as secondary messengers, beta-oxidation in peroxisomes, prostaglandin synthesis (lipoxygenases and cyclooxygenases) and cytochrome P450 detoxification reactions (Boonstra & Post, 2004).

During development, ROS have a critical function as regulators of the mitotic cell cycle resulting in 4 possible outcomes: cell cycle induction, cell cycle arrest for repair or for permanent termination, cellular differentiation, or apoptosis (Boonstra & Post, 2004). Different cellular concentrations of ROS can lead to progression or termination of the cell cycle. Low ROS conditions will favor growth-factor stimulated pathways resulting in cell cycle progression, but if

6 prolonged, low ROS can result in differentiation-like growth arrest (Markovic et al., 2007). To maintain a low state of ROS during proliferation, endogenous antioxidant enzymes such as glutathione (GSH) have been shown to be recruited into the nucleus (Markovic et al., 2007).

Nucleotide erythroid like factor 2 like 2 (Nrf2) is a transcription factor that is activated by

H2O2, and induces the activation of the antioxidant response element (ARE) genes (Sussan et al.,

2017). In response, proteins that favor cell survival are expressed, including: antioxidant enzymes, proteins favoring glutathione synthesis and regeneration, clearing of oxidized proteins, repair of damaged DNA, and anti-inflammatory genes (Sussan et al., 2017). Nrf2 is only transiently active and is quickly tagged for degradation. The transient nature of Nrf2 action suggests that prolonged activation is detrimental to the cell, possibly through reductive damage

(Flohe & Flohe, 2011).

1.2.2 Antioxidant Enzymes

•- Superoxide dismutase (SOD) rapidly converts superoxide free radical (O2 ) to water and

hydrogen peroxide (H2O2). SOD-1 requires a manganese metal cofactor, whereas SOD-2

requires copper or zinc metal cofactor (Vlamis-Gardikas & Holmgren, 1989). H2O2 is less toxic

•- to the cell than O2 , but is much more abundant. H2O2 is not considered a free radical, which is a

molecule with an unpaired electron. H2O2 is rather considered a reactive oxygen species (ROS) since in the presence of transition metals, is converted to OH- which is a free radical (Vlamis-

Gardikas & Holmgren, 1989). Thus, catalase (CAT), glutathione peroxidase (GPx), and peroxiredoxin (Prdx) all catalyze the conversion of H2O2 to water. Thioredoxin (Txn) also acts as an ROS and free radical scavenger, catalyzing many reactions, including: reduction of protein

7 disulfide bonds, redox regulation of enzymes and transcription factors (Vlamis-Gardikas &

Holmgren, 1989). Particularly, Trx acts as a cofactor for the activation of glucocorticoid receptor and subsequent binding of glucocorticoids (Makino et al., 1999).

1.2.3 Oxidative damage (markers)

As the concentration and length of exposure to ROS increases, the cell is less able to cope with these toxic substances with endogenous antioxidant mechanisms, resulting in oxidative stress. ROS react strongly with all classes of biomolecules through lipid peroxidation, protein carbonylation, and DNA peroxidation. Free radicals react with polyunsaturated fatty acids

(PUFA) in a self-perpetuating manner that results in the production of lipid hydroperoxides, free radical species, and aldehydes (e.g. malondialdehyde [MDA] and 4-hydroxynonenal [4-HNE]).

The self-perpetuating nature of lipid peroxidation and aldehydic by-products spread damage to other parts of the cell including protein and DNA (Mates, 2000; Nimse et al., 2015). Oxidative damage to proteins leads to degradation when free radicals and trace metal ions such as Fe2+ or

Cu2+ react with amino acid residues. The most susceptible amino acid residues are lysine, proline, histidine, and arginine. The carbonyl residue generated by these processes is a useful measure of oxidative damage of proteins and can be detected by spectrophotometry (Nimse et al., 2015). The free hydroxyl radical (OH-) is especially reactive and causes oxidative damage to

DNA by cleaving one strand of the helix at the guanine residues of cytosine-phosphate-guanine

(CpG) dinucleotide, causing the supercoiled DNA to unwind (Nimse et al., 2015). The resulting

8-hydroxyguanine (8-OH-G) lesion is a common marker of DNA peroxidation resulting from oxidative damage and can be detected through an enzyme-linked immunosorbent assay (ELISA)

(Wells et al., 2009). Please see Figure 1.1 for a diagram of cellular oxidative stress.

8

1.2.4 Oxidative stress progression

Oxidative DNA damage leads to initiation of damage repair pathways, one of which is through the activation of p53 protein. Under low stress conditions, small increases in p53 activity have been associated with decreased ROS production, cell cycle arrest, and lower mitochondrial activity (Vousden et al., 2009). If the elevated ROS levels are sustained or increase further, this increases p53 activity, one of the key signals that leads to initiation of apoptotic cascades (Wells et al., 2009).

Reactive oxygen species (ROS) have hormetic properties: excessive levels of ROS are associated with cellular damage and overall pathology, whereas smaller deviations from homeostasis activates cellular signaling through transcriptional activation. It has been suggested that most, if not all pathways resulting in transcriptional activation are regulated by oxidative signals (Flohe & Flohe, 2011). It has been proposed that as ROS accumulates in the cell, cellular signaling pathways are triggered: low levels of mitochondrial ROS are required for physiologically normal cell cycle events such as proliferation/ differentiation. Further increase in ROS production leads to expression of adaptive genes including antioxidant enzymes. When the intracellular oxidative potential overcomes the antioxidant capacity of the cell, senescence and apoptosis is triggered and leads to cell-wide oxidative damage (Hamanaka & Chandel,

2010).

Therefore, the tight regulation of ROS levels is essential to guide cell fate, particularly during development. During early development, the fetus maintains a preferentially reductive

9 environment which favors proliferation. Later in development, ROS levels rise to accommodate differentiation (Wells et al., 2009). Overproduction of cellular ROS can occur in response to endogenous factors such as extracellular matrix factors and growth factors, or exogenous factors such as xenobiotics. Endogenous enzymatic and non-enzymatic antioxidant mechanism are in place to control ROS production and prevent spread of oxidative damage to major macromolecules. Mitochondria-specific isoforms of these enzymes regulate the major site of

ROS production (Apostolova et al., 2015).

1.3.0 Clinical significance of oxidative damage in utero

ROS regulates cell fate and cellular signaling and thus, is important for normal developmental processes such as proliferation forming the blastocyst, neuronal differentiation, and formation of digits (Dennery, 2007). Inappropriate changes or imbalances in the cellular redox state can lead to oxidative damage and in extreme cases, apoptosis and necrosis. It is important to understand the oxidative changes in normal pregnancy, how this may differ in complicated pregnancy, and the impacts on the fetus and later life of the child.

1.3.1 Normal pregnancy – baseline ROS

Normal pregnancy is associated with changes in oxidative potential (Toescu et al., 2002).

A small study comparing pregnant mothers (n=17) and non-pregnant women (n=12) found that pregnancy was associated with an initial decrease in maternal venous total antioxidant capacity

(TAC) in the first trimester that gradually increased over gestation and exceeded control levels

10 up to 8 weeks post-partum (Toescu et al., 2002). Another study compared the total antioxidant capacity of healthy children delivered at term. At the time of birth, female offspring had a higher

TAC, as well as, increased activity of superoxide dismutase, glutathione peroxidase, and catalase than males. This would suggest female offspring are better able to deal with the hyperoxic delivery event than male offspring (Diaz-Castro et al., 2016). Measures of urinary 8-OHdG in the first trimester of pregnant controls have been found to exceed that of non-pregnant women

(Potdar et al., 2009). Finally, a longitudinal study in healthy full-term neonates followed infants for 1, 3.5, 6 and 12 months postnatally. Enzymatic activity of superoxide dismutase (SOD) and catalase peaked at 3.5 months, and gradually decreased, leveling off at 1 year of age. At 1 year,

SOD was still significantly increased compared to average adult values, suggesting that infants experience oxidative stress early in life (Friel et al., 2004).

1.3.2 ROS leading to pregnancy complications

Complicated pregnancies including diabetes, preeclampsia, IUGR, have been associated with oxidative stress (Potdar et al., 2009). One relatively large, and controlled study established an association between increased urinary and plasma markers of oxidative stress during late second trimester (24-26 weeks gestation) and later pregnancy complications including preeclampsia, small for gestational age babies, or low birthweight babies. They controlled for demographic factors such as maternal age, parity, pre-pregnancy body-mass index, fetal gender, previous preterm birth, and conception methods, suggesting that oxidative stress is an important factor in the pathogenesis of these complications in otherwise healthy singleton pregnancies

(Hsieh et al., 2012). Conflicting evidence was found with a study that showed increased maternal urinary 8-OHDG to be associated with a lower incidence of preterm birth compared to

11 healthy term pregnancy (Ferguson et al., 2015). Another study suggested that oxidative stress levels in the amniotic fluid are directly relevant to the oxidative status of the offspring, as they are in immediate contact with the fetus. One study measured oxidative stress markers in the amniotic fluid at the end of the first trimester (16-19 wks gestation) and the association with pregnancy complications, including gestational diabetes mellitus, pregnancy-induced hypertension, and bacterial vaginosis (Bogavac et al., 2012). While serum concentrations of antioxidant enzymes are more informative, they found that there was a significant decrease in the lipid peroxidation by-product, malondialdehyde in complicated pregnancies compared to controls.

While the relationship between increased ROS and pregnancy complications might result from an imbalance in gestational oxidative homeostasis, there have been many studies to show the association between increased ROS levels and adverse fetal outcome. Low levels of ROS are particularly important early in gestation (<20 wks gestation) for proliferation. One study found polymorphisms in four key genes regulated by the antioxidant response element (GPx4, ABCB1,

COMT, and OGG1) in systemic maternal blood samples contributed significantly to an increased risk of idiopathic recurrent miscarriage, independent of all other lifestyle factors (Khadzhieva et al., 2014). It has also been shown that increased maternal urinary 8-OH-dG at 12 and 28 weeks of gestation is associated with increased incidence of small-for gestational age babies, suggesting this might be indicative of early oxidative changes in placenta (Potdar et al., 2009).

12 1.3.3 Long-term outcomes later in the life of the child

Oxidative insult during the fetal period not only risks the health of the newborn in the perinatal period, but it can also lead to adverse long-term neurodevelopmental, cardiac, and metabolic outcomes.

Prenatal oxidative insult has been associated with later neurobehavioural problems, including ADHD, schizophrenia, and anxiety and depression like behaviours. One study found that prenatal ethanol exposure led to oxidative stress in young rats, with increased antioxidant enzyme activity and depleted levels of total glutathione (Brocardo et al., 2012). Exposure to ethanol and tobacco in utero has not only been associated with adverse neurodevelopmental and behavioural changes, but these effects were mediated through an oxidative stress mechanism (Li et al., 2004). There was a significant decrease in the activity of SOD, GPX, and CAT antioxidant enzymes, and increased malondialdehyde content in both the liver and brain of treated mice offspring (Li et al., 2004). Bipolar patients have been shown to have lower levels of glutathione- s-transferase (GST) in the prefrontal cortex (Gawryluk et al., 2011). 3 months following administration of selective serotonin reuptake inhibitors to patients with major depressive disorder, levels of malondialdehyde were decreased and the activity of several antioxidant enzymes was normalized. (Bilici et al., 2001; Balsmus et al., 2015). These findings suggest that oxidative stress may play a role in modulating these mood disorders. By priming the unborn child to be more responsive to oxidative stress later in life, stressors that would normally be small or inconsequential can have significant adverse effects particularly in the brain, contributing to the development of mood disorders.

13 Early hypoxic insult has also been associated with adverse cardiac outcomes, including an increased risk for later cardiovascular disease. One study used a rodent model of fetal hypoxia-induced IUGR, where hypoxic insult was administered at 12% oxygen from day 15-22 of pregnancy. The offspring were observed 4 months and 7 months postnatally, and were found to have increased cardiac remodelling, with decreased expression and activity of matrix metalloproteinases and actin proteins. They also found an increase in collagen 1 and 3 in the left ventricle of the hypoxic injury group at both 4 and 7 months postnatally. The hypoxia induced

IUGR group displayed significantly reduced cardiac function at 4 months, and at 7 months, the offspring had reduced cardiac output (Xu et al., 2006). These early adverse cardiac outcomes might persist into adulthood. One study considered the development of atherosclerosis in adulthood following in utero hypoxic insult in a rodent model. From days 7-21 of pregnancy, pregnant rats were housed in 10% oxygen for 3 hours/day resulting in IUGR pups. At 12 months of age, the hypoxia induced IUGR offspring were exposed to hypoxic challenge of two, 4 hour periods per day, for 4 weeks. The hypoxia-induced IUGR pups exhibited catch-up growth, and there were no differences in total body weight or individual organ weights at PND28. At 16 months, cardiac function was measured and suggested maternal hypoxia led to increased aortic lesions similar to early atherosclerosis, thickening of the internal aortic wall, inflammation of the muscle cells, as well as invasion of smooth muscle cells into the internal aortic wall (Wang et al.,

2009). They found that secondary hypoxic challenge did not exacerbate the problem, but that overall, maternal hypoxia led to early signs of atherosclerosis in adult offspring (Wang et al.,

2009). Work by Giussani et al. found that in utero hypoxia-induced adverse long-term cardiac outcomes could be prevented through antioxidant therapy. In a rat model of maternal hypoxia, they showed parallel ascorbic acid (vitamin C) treatment (5mg/ml in drinking water) prevented

14 aortic wall thickening at birth (Giussani et al., 2012). At 4 months, while the morphology of the aorta in adult offspring of maternal hypoxia resolved, they had a lower functional cardiac response, whereas antioxidant treatment was able to reverse this effect (Giussani et al., 2012).

This suggests that after antioxidant treatment, cardiac outcomes of maternal hypoxia can be reversed and that secondary hypoxic insult does not further exacerbate the cardiac damage.

Early hypoxic insult has also been associated with adverse metabolic outcomes, particularly affecting the liver. A study by Wang et al. measured the relative organ weights of newborn pups following maternal hypoxia and found the liver to be significantly smaller than controls (Wang et al., 2009). One longitudinal study examined metabolic outcomes in neonates and adult offspring following a similar model of maternal hypoxia. Increased blood lipid and insulin were observed in neonatal. Higher levels of PEPCK, a gluconeogenesis-associated protein, as well as an increase in the insulin regulation proteins: INSR-b, AKT, and Glut-2 were also observed. Maternal hypoxia in pregnancy resulted in increased blood lipid, but no difference in fasting glucose or insulin in adult offspring compared to offspring from normoxic pregnancies. PEPCK protein expression remained increased in the adult offspring, whereas the expression of factors involved in insulin regulation including IRS-2, AKT, and Glut-2 were significantly decreased compared to their age-matched controls (You et al., 2016). Nrf2 is a transcription factor for the antioxidant response element (ARE) which activates transcription of antioxidant enzymes. One study investigated both in vitro and in vivo activation of Nrf2, and outcomes on the progression of non-alcoholic fatty acid liver disease (NAFLD). They showed that activation of Nrf2 through two independent mechanisms prevented hepatic fibrosis when

15 rats were fed with a choline-deficient l-amino acid defined (CDAA) diet which promotes

NAFLD (Shimozono et al., 2013).

1.4.0 Establishing mechanism between GC and oxidative stress – in vitro

Antenatal synthetic glucocorticoid therapy has been shown to be associated with long- term adverse fetal outcomes. Oxidative stress might be one mechanism through which this might occur. Acute oxidative stress has been associated with acute synthetic glucocorticoid exposure in vivo and in vitro.

One in vitro study investigated a direct association between synthetic glucocorticoid exposure and oxidative stress. Using organotypic hippocampal slice cultures, sGC exposure (50

µmol/L) increased ROS production from 2 to 6 hours, peaking at 4 hours following initial exposure and returning to baseline 8 hours following exposure. Glucocorticoid treatment also led to a slight decrease in expression of the antioxidant, glutathione peroxidase, and an increase in the expression of NADPH oxidase at 4 hours (You et al., 2009). This study clearly establishes

ROS production in response to glucocorticoid signaling in the hippocampal cells.

Alam et al. examined the interaction between glucocorticoid receptors and the antioxidant response element (ARE) transcription factor, Nrf2 in the rat liver. In a series of in vitro and in vivo experiments, it was shown that following the dexamethasone-activation of the glucocorticoid receptor, Nrf2 is prevented from binding the ARE enhancer site through histone deacetylation at H3K27. Further, this was associated with decreased gene expression of

16 downstream targets of ARE; NADPH quinone dehydrogenase 1 (NQO1), glucose-6-phosphate dehydrogenase (G6Pdx), as well as glutamate-cysteine ligase modifier subunit (GCLM) (Alam et al., 2017). This would suggest that there is a direct mechanistic inhibition of the ARE following glucocorticoid receptor activation. Taken together, these studies indicate that glucocorticoids can induce cellular ROS while mechanistically inhibiting the cell’s ability to mount an antioxidant response.

This combination of glucocorticoid-mediated induction of ROS, as well as a blunted antioxidant response further suggests that there is increased oxidative damage through protein carbonylation, lipid peroxidation, and/or DNA peroxidation. Chronic glucocorticoid exposure has been demonstrated to increase protein carbonylation and decrease mitochondrial function in a rat adrenal medulla cell line (rat pheochromocytoma PC12 cells; Tang et al., 2013). Exposure to various concentrations of corticosterone administration for 24 hours led to increased protein carbonylation, decreased activity of mitochondrial complex 1, and decreased SOD activity (Tang et al., 2013). Interestingly, while glucocorticoid exposure increases cellular ROS, mitochondrial activity was decreased following treatment.

One study showed that sGC induced oxidative damage in neuro-epithelial like pluripotent stem cells, and that the antioxidant, N-acetylcysteine, was able to reverse this (Raciti et al.,

2016). The authors directly exposed one set of cells to dexamethasone and derived a secondary set from the first. They observed an increase in ROS production and a decrease in the gene expression of the antioxidant enzymes, CAT, SOD1, SOD2, and GPX7 in the cells which were never directly exposed to dexamethasone. The study also identified a decrease in differentiation in the daughter cells, with a decrease in the neuronal markers, vGLUT2, GAD67, MAP2, and

DCX. However, the daughter cells expressed an increase in the glial marker, GFAP, suggesting

17 that glucocorticoid exposure might decrease the likelihood of neurogenesis through a mechanism that affects differentiation. Subsequent treatment with the antioxidant NAC prevented this increased expression of GFAP, and rescued the expression of the neuronal markers in daughter cells, suggesting that oxidative mechanisms might be regulating this inhibition of neurogenesis following distant glucocorticoid exposure. As cell fate and cell cycle regulation is heavily dependent on cellular ROS signaling (Boonstra & Post, 2004), early (in utero) glucocorticoid exposure might fundamentally alter the ROS homeostasis of neural stem cells, thus altering neuronal differentiation outcomes of adult neurogenesis.

1.5.0 Effects of GC on oxidative stress, in vivo

To establish effects of glucocorticoids on basal antioxidant enzyme activity in vivo,

McIntosh et al. (1998) exposed rats to either subcutaneous injections of corticosterone (10 mg/kg) or adrenalectomy to remove endogenous glucocorticoid synthesis. The authors then compared the enzyme activity of Cu/Zn SOD, Mn SOD, GPx, and CAT. They found Cu/Zn

SOD activity to be significantly more active in the brain, but less active in the liver following glucocorticoid treatment, compared to adrenalectomized controls; however, there was no difference in MnSOD activity. GPx activity was downregulated in the hippocampus, cortex, and liver following glucocorticoid treatment, but CAT was only downregulated in the liver

(McIntosh et al., 1998). These results suggest glucocorticoids have a regulatory role on basal antioxidant enzyme activity, not only transcriptional and translational expression as seen in previous studies.

18 Zafir & Banu investigated the effects of in vivo glucocorticoid exposure on systemic oxidative stress, focusing on the brain, heart, and liver in adult rodents. They administered 10,

20, and 40 mg/kg corticosterone orally to rodents for 21 days. They found that chronic glucocorticoid exposure led to significantly lower activity of the antioxidant enzymes, SOD,

CAT, GST, and GR. They also found significantly increased measures of protein carbonylation, as well as, malondialdehyde, in all three tissues (Zafir & Banu, 2008). While the doses used in this study were high, the study does suggest that chronic glucocorticoid exposure can induce systemic oxidative damage in tissues relevant to long-term adverse outcomes of increased oxidative stress. Alternatively, these results could have been a product of the toxic doses of GC administered, but further work would need to be done to address this possibility.

Sato et al. administered a physiologically relevant dose of corticosterone (0.5 mg/kg) for

14 days to adult rats to investigate whether chronically increased HPA activity could lead to oxidative damage in the brain. Glucocorticoid treated rats had higher hippocampal lipid hydroperoxides as well as increased protein carbonylation (Sato et al., 2010). In addition, these animals exhibited significantly lower hippocampal antioxidant enzyme activity of SOD, CAT and GPx (Sato et al., 2010). The Morris water maze was used as a measure of learning and memory, suggesting that chronic corticosterone exposure did not impact learning, but led to significantly impaired memory performance 5 days following learning. This suggests that not only are glucocorticoids inducing altered oxidative stress in the hippocampus, but that this is also associated with impaired memory performance.

Interestingly, in the three studies described above, there was an increase in oxidative protein damage and an associated decrease in antioxidant enzyme activity. This could be due to direct oxidative damage on the antioxidant enzymes, leading to less functional protein. One

19 study investigated the mechanism behind glucocorticoid-induced decrease of GPx enzyme activity. In an in vitro model of E18 fetal rat hippocampal cultures, they found corticosterone administration decreased levels of glutathione (Patel et al., 2002). Glutathione is an endogenous non-enzymatic antioxidant and is the ligand for GPx. 90% of cellular glutathione is in a reduced form, and during oxidative stress, is oxidized to a glutathione dimer through a disulfide bond

(GSSG). As corticosterone decreased the overall cellular level of GSH, a decrease in ligand would decrease the enzyme kinetics of GPx. Not surprisingly, they did also observe a decrease in the GPx enzyme activity following glucocorticoid exposure (Patel et al., 2002).

1.6.0 Effects of prenatal GC on oxidative stress in fetus/ later outcomes in life

With a well-established link between glucocorticoids and oxidative stress outcomes in vitro and in vivo adult animal models, it is critical to understand the implications of sGC therapy on oxidative stress in the fetus. One study examined cord blood levels of oxidative stress markers following betamethasone. In umbilical vein plasma, malondialdehyde was significantly increased in samples collected within 72 hours after the last betamethasone exposure, compared to samples collected beyond 72 hours following the last betamethasone exposure. GPx3 activity was decreased in samples collected 24 hours after last betamethasone exposure (Verhaeghe et al.,

2009).

Stark et al. studied the effect of timing of prenatal glucocorticoid exposure on oxidative stress outcomes in the human placenta. The placenta is glucocorticoid sensitive, showing a

20 downregulation of 11-BHSD2 in only female offspring following betamethasone exposure (Stark et al., 2009). There was an effect of betamethasone on both sex and timing of exposure. Human placentae were collected from mothers given sGC and who delivered either within 72 hours or after 72 hours. They found that male placentae were more susceptible to oxidative damage than female placentae, with higher levels of protein carbonylation, lipid hydroperoxides, and nitrotyrosine content, irrespective of betamethasone timing. These male placentae also showed lower GPx3 activity than time-matched female placentae. When measuring oxidative stress in placentae from offspring treated with sGC and delivered 72 hours after last exposure, the placentae samples from the male offspring continued to display more oxidative damage than females, through increased protein carbonylation and lower GPx3 activity. However, when placentae were collected beyond 72 hours following last sGC exposure, there were no sex- specific changes seen in lipid peroxidation and nitrotyrosine content (Stark et al., 2011). Thus, since the placenta is the main interface between the mother and the child, sGC-induced oxidative stress to this organ might endanger the developing fetus.

Redox changes in the placenta have been associated with increased risk of developing pregnancy complications, as discussed earlier. Therefore, it is important to determine whether glucocorticoid exposure can induce any changes to the placenta in a redox-sensitive mechanism.

One study investigated juvenile offspring of marmoset monkeys treated with sGC in utero and showed that there was an increase in antioxidant enzyme gene expression in the aorta at 2 years of age (Atanasova et al., 2009). The pregnant animals were treated at two time points with oral dexamethasone (5 mg/kg; 7 days), early in gestation and late in gestation; only late gestation exposure resulted in significant changes. GC exposure resulted in increased expression of GPx1,

SOD2, GCLM, and GSR and a trend towards increased expression of GPx4. Urinary F-

21 isoprostanes were also investigated as a marker of lipid peroxidation at 1, 6, 12, and 24 months, though no changes were identified compared to controls. This study suggests in utero sGC- induced programming of antioxidant enzymes in the aorta. These effects appear to be tissue- specific and enzyme-specific, where the same treatment (prenatal dexamethasone) can cause upregulation of catalase (CAT) gene expression in some tissues (lung, renal cortex; Chen et al.,

2004), downregulation in others (cerebellar cortex; Ahlbom et al., 2000) and no change in others, such as the aorta (Atanasova et al., 2009). It is therefore important to investigate the expression of each individual antioxidant enzyme in different fetal tissues to elucidate their role during gestation and in response to sGC treatment.

22 Figure 1.1 – Cellular Oxidative Stress

Figure 1.1 - ROS and RNS are produced as by-products of normal cellular reactions. Endogenous antioxidant systems act to maintain cellular redox homeostasis. Small changes in this cellular redox state can lead to important cellular signalling, such as for proliferation or differentiation. However, prolonged and/or severe imbalance of cellular ROS balance can lead to oxidative damage through DNA, protein, and lipid peroxidation. Lipid peroxidation is particularly damaging as the aldehydic by-products, 4-HNE as well as MDA can propagate protein carbonylation as well as DNA peroxidation. ROS reacting with DNA can produce 8- hydroxy-2'-deoxyguanosine, an oxidized guanine species. This can act as a lesion that prevents DNA methyl transferase from binding and can produce altered epigenetic patterns.

23 2. Objective, Rationale, and Hypothesis

2.1 Objective:

To investigate the effects of prenatal synthetic glucocorticoids (sGC) on the oxidative state of fetal hippocampus, placenta, and liver through quantification of antioxidant enzyme mRNA expression, protein carbonylation, glutathione redox ratio, and lipid peroxidation.

2.2 Rationale:

2.2.1 Brain – Hippocampus

Within the brain, the hippocampus is particularly sensitive to the effects of sGC therapy due to the high density of glucocorticoid receptors (Matthews et al., 1998). The brain is also particularly susceptible to oxidative damage due to its high metabolic activity, low antioxidant capacity, and relatively high lipid content (Ostrakhovitch et al., 2013).

Prenatal administration of betamethasone lead to increased lipid oxidative damage

(MDA), and a decrease in the concentration of GPx-3 in human umbilical vein plasma

(Verhaeghe et al., 2009). Maternal restraint stress in late gestation lead to an increase in oxidative mitochondrial DNA damage (8-OH-dG lesion) in the hippocampus of rat offspring

(Song et al., 2009). Furthermore, a study in adult rodents showed chronic administration of sGC

(0.5 mg/kg daily for 14 days) to be associated with a significant reduction in hippocampal activity of antioxidant enzymes, and a significant increase in the levels of protein carbonylation.

24 Therefore, sGC does have a direct negative oxidative impact on the brain and the current study focused on the hippocampus, a key region of sGC programming.

2.2.2 Placenta

The placenta has multiple functions in relation to fetal development, including: nutrient and gas transport, secretion of hormones, and a selective barrier. Different stressors including maternal nutrient restriction, hypoxia, and maternal stress can impact these functions differently

(Burton et al., 2016).

The placenta was investigated as it is the primary interface between the maternal and fetal circulation, and is a glucocorticoid-sensitive organ. Administration of dexamethasone in mice during late gestation lead to lower placental weight (Baisden et al., 2007). Another study showed that prenatal administration of betamethasone lead to increased 11b-HSD2 activity in human female placenta, when measured within 72 hours. This suggests that sGC exposure impacts the placental barrier to GC in a sex and time-dependent manner (Stark et al., 2009).

Additionally, prenatal sGC exposure in humans showed a strong sex-dependent placental oxidative stress profile : male placentae collected within 72 hours of betamethasone treatment had significantly higher levels of protein carbonylation, lipid hydroperoxides, nitrotyrosine levels, and reduced glutathione peroxidase (GPx-3) activity when compared to female placentae delivered at the same time point (Stark et al., 2011). When delivered beyond 72 hours following betamethasone exposure, the male placentae consistently showed elevated protein carbonylation levels, and reduced GPx-3 activity when compared to female placentae delivered at the same time point (Stark et al., 2011).

25

2.2.3 Fetal Liver

The adult liver has primarily 3 functions: to filter blood of toxins, to metabolize amino acids and lipids, and gluconeogenesis. On the other hand, the fetal liver primarily functions as the main hematopoetic organ during mid-late gestation and has significant roles in protein and lipid synthesis (Kamiya et al., 2003; Dawkins, 1966). During the perinatal period, the fetal liver begins to express enzymes required for xenobiotic clearance, and close to term, the fetal liver begins to prepare for gluconeogenesis (Fowden et al., 2016).

Rats exposed to sGC in late-gestation showed life-long elevations in hepatic expression of the glucocorticoid receptor (GR) and phosphoenolpyruvate carboxykinase (PEPCK), the rate- limiting enzyme of gluconeogenesis (Nyirenda et al., 1998). In sheep, multiple-course prenatal sGC administration lead to altered glucocorticoid metabolism in the fetal liver through increased

11B-HSD1 mRNA and protein expression at gestational day 125 (term ~ 147 days), but not at term (Sloboda et al., 2002).

Prenatal glucocorticoid exposure has been associated with reduced birth weight.

Associated metabolic outcomes later in life include insulin dysregulation and predisposition to non-alcoholic fatty liver disease (NAFLD) (Zhang et al., 2014). In rodents, prenatal glucocorticoid over-exposure lead to reduced birth weight and increased lipid accumulation in offspring liver (Drake et al., 2010). Following 6 months on a high-fat diet, animals exposed to sGC in utero showed changes in gene expression related to the development of NAFLD (Drake et al., 2010).

26

A meta-analysis of studies investigating oxidative stress following chronic glucocorticoid exposure in adult animals showed that following the brain, the liver was the organ with the second highest effect size (Costantini et al., 2011). In adult guinea pig, diet-induced lipid dysregulation lead to increased MDA levels and reduced SOD activity (Tveden-Nyborg et al., 2016).

Therefore, sGC therapy is administered to the fetus when the developing liver is responsive to glucocorticoids, at a developmental time point when lipid metabolism is being programmed, of which dysregulation can lead to oxidative stress. No study has yet looked at synthetic glucocorticoid-induced hepatic oxidative stress in fetal life. In the current study, the fetal liver was investigated as it expresses a high density of glucocorticoid receptors and is the major site of glutathione production, a key tissue antioxidant (Sloboda et al., 2002; Pinto &

Bartley, 1969).

2.3 Hypothesis

Prenatal sGC therapy will increase markers of oxidative stress in the fetal hippocampus, placenta, and liver, through an increase in the gene expression of one or more enzymes from the antioxidant defense mechanism, increased protein carbonylation, and an increased ratio of oxidized to reduced glutathione.

27

3. Methods

3.1 Animal protocol

The guinea pig was used as the gestational model for several reasons. Firstly, the guinea pig has a similar neurodevelopmental profile to humans, in utero. Secondly, guinea pig give birth to neuroanatomically mature young. Additionally, guinea pigs have longer gestation times compared to other rodent models. Finally, similar to humans, guinea pig produce cortisol as their primary glucocorticoid, as opposed to other rodents which produce corticosterone (Dobbing

& Sands, 1979).

All animal work was performed by Alex Mouratidis. 12-week old virgin female guinea pigs were obtained from Charles River Canada and were housed individually in 12 hour light/dark cycles. Animals had access to food and water ad libidum. Animals were bred as we have described previously (Dean & Matthews, 1999). On GD40,41 as well as GD50,51, the pregnant animals were given subcutaneous injections of either betamethasone (1 mg/kg), or an equal volume of saline (0.166ml/kg). The betamethasone dose was based on current doses prescribed to mothers at risk of preterm birth (0.25 mg/kg). However, guinea pig glucocorticoid receptors have a 4-fold lower affinity for sGCs and thus, the dose was adjusted accordingly

(Crudo et al., 2013).

Animals were euthanized on GD52, 24 hours following the final exposure to betamethasone. Fetal and maternal hippocampal, pituitary, adrenal, liver, gonadal, and placental

28 tissues were collected and immediately frozen on dry ice and stored at -80°C until further processing. Of these tissues, fetal hippocampus, liver, and placenta, as well as maternal hippocampus was analyzed for this project. Only one female and male offspring per litter was selected randomly for analysis. This yielded 4 groups: female veh (n=8), female beta (n=10), male veh (n=5), and male beta (n=8). The animal protocol was approved by the Animal Care

Committee at the University of Toronto in accordance with the Canadian Council for Animal

Care.

3.2 Quantitative Real-Time PCR (qRT-PCR)

3.2.1 Primer design, standards, efficiency

Target genes were based on the literature: antioxidant enzyme genes are downstream of the antioxidant response element (ARE) which is activated by the nuclear-factor E2 p45 related factor 2 (Nrf2) following oxidative stress (Malhotra et al., 2010; Hayes et al., 2014; Ma, 2013).

Primers were designed using NCBI primer blast software. Suggested primers were tested on oligocalc.org for potential hairpin structures prior to ordering from Integrated DNA

Technologies (Coralville, Iowa, USA).

Primers were reconstituted using RNAse/DNAse-free water to a stock concentration of

100 µM. Stock primers were diluted to a 1:9 working dilution using RNAse/DNAse-free water.

Standard curves were run to test the reaction efficiency of the primers. Standards were run using cDNA concentrations of: 100 ng/µL, 10 ng/µL, 1 ng/µL, 0.1 ng/µL, and 0.01 ng/µL. Primers

29 with an efficiency reading between 90-110% were considered appropriate for use. Primers were used at a concentration of cDNA where the cycle threshold (Ct) fell below 32.

3.2.2 Tissue preparation, RNA extraction and cDNA conversion

Tissues were homogenized on dry ice using liquid nitrogen and a chilled mortar and pestle. Random ID’s were assigned at this point using a random number generator. Males and females were run and analyzed separately. Tissue (30mg) was homogenized in a 1:10 solution of betamercaptoethanol and RLT buffer (600µL) using a chilled steel ball at 25 Hz for 1 min. RNA was subsequently extracted using the commercially available kit, AllPrep DNA/RNA/miRNA

Universal Kit (Qiagen, Hilden, DEU), following the manufacturers protocol. RNA integrity was evaluated through agarose gel, and RNA purity and concentration was evaluated using a

Nanodrop 1000 Spectrophotometer (Fisher Scientific, Hampton, NH, USA). A 260/280 value of

2 was used as a reliable marker of RNA purity. RNA integrity was evaluated through agarose gel electrophoresis, by the presence of 28S and 18S rRNA bands. RNA was stored in

RNAse/DNAse-free water at -80°C until further use.

RNA was thawed on wet ice prior to use for cDNA conversion. For each sample conversion, SensiFAST cDNA synthesis kit (5µL; Bioline; London, GBR) was used, composed of a mix of of Transamp Buffer (4µL; Bioline) and reverse transcriptase (1uL; Bioline). Based on the RNA concentration, a final reaction volume (20µL) was made up with RNAse/DNAse- free water. One RNA sample was randomly selected to run a negative control, omitting the reverse transcriptase from the SensiFAST mix and replacing it with RNAse/DNAse-free water.

The cDNA conversion was completed through the CFX 96 Real-Time system C1000 Thermal

30 Cycler (Bio-Rad Laboratories; Hercules, CA, USA). The concentration of the resulting stock cDNA was 50 ng/µL.

3.2.3 PCR, housekeeping genes, list of genes

The mRNA expression for target genes was quantified by quantitative real-time PCR

(qRT-PCR), using SensiFAST Sybr HI-Rox Mix (Bioline; London, GBR) and the Bio-Rad

CFX96 Touch Real-Time PCR Detection System (Bio-Rad; CA, USA). Please see Table 3.1 for a list of primer sequences used. Data was analyzed using CFX Manager Software (Bio-Rad).

A mastermix was made up of SensiFAST (Bioline; London, GBR), water and the forward and reverse primers (Integrated DNA Technologies; Coralville, IA, USA) to a final volume of 18

µL per reaction in a ratio of 10:6.4:0.8:0.8 respectively. The stock cDNA was diluted to a working concentration (4 ng/µL), of which 2 µL was added to the reaction. All genes were run at this concentration. Each PCR plate was run with a negative no-template control using water.

The negative reverse transcriptase control from the cDNA conversion was run once to ensure cDNA purity. Each sample was run in triplicate, and were included if the standard deviation between the replicates was under 0.3. If the standard deviation fell above 0.3 amongst the triplicates, the furthest sample read was excluded from the analysis. Samples were compared to the geometric mean of the two housekeeping genes: Gapdh and Ywhaz. Please see figures 3.1,

3.2, 3.3, and 3.4 for housekeeping gene stability. Calculations for the relative mRNA expression were analyzed using the 2-ΔΔCt method.

31 3.3 Protein Carbonylation

3.3.1 Protein Extraction, storage times/conditions

Protein was extracted using RIPA lysis buffer (Cell Signaling Technology; Danvers, MA,

USA). 1x RIPA lysis buffer was prepared using 9 mL RNAse/DNAse-free water and 1mL 10x

RIPA lysis buffer. One protease inhibitor tablet was added per 10 mL RIPA lysis buffer, and was mixed by inverting. RIPA lysis buffer was stored at 4°C for up to two weeks. 300 µL RIPA was added to 30 mg of powdered placenta tissue and 500 µL RIPA was added to 30 mg of powdered liver tissue. Hippocampal samples were not analyzed due to limited availability of sample tissue. A chilled steel ball was added to the eppendorf tube and homogenized in chilled homogenizing plates (1 min at 25Hz). Homogenized samples were allowed to rest on wet ice to settle foam, and then centrifuged (13500 RPM, 15 min, 4°C). The supernatant was collected and stored at -80°C.

3.3.2 Bradford

To quantify protein content following extraction, a standard Bradford assay was performed. A sample of bovine serum albumin (BSA, 1 mg/mL; Sigma-Aldrich, St Louis, MO,

USA) which was stored at -20°C was thawed and diluted to 7 standards using RNAse/DNAse- free water. The standard range was between 0-25 µg/µL, and 1 µL of each sample was prepared using standard Bradford Reagent (Sigma-Aldrich; St. Louis, MO, USA) to quantify protein.

Samples were diluted with 1x RIPA buffer until they fell within this range.

3.3.3 Protein carbonylation

32 To measure protein carbonylation in protein extracts, the Protein Carbonyl Content Assay

Kit (Ab126287, Abcam; Cambridge, GBR) was used. Protein carbonyl groups are reacted with

2,4-dinitrodiphenylhydrazine (DNPH, supplied), and the resulting 2,4-dinitrophenylhydrazone

(DNP hydrazone) is quantified with spectrophotometry at 375 nm. Upon receipt, the kit was stored at 4°C away from light. Prior to running the assay, DNPH solution, 10% streptozocin

(supplied), and 6M guanidine (supplied) were brought to room temperature, whereas 100% trichloroacetic acid (TCA, supplied) was kept on ice. Using RNAse/DNAse-free water, protein samples were diluted to 200 µL aliquots at an optimal concentration of 7 µg/µL for this assay.

Acetone (10 mL; not supplied, Caledon Laboratories, Georgetown, ON, Canada) was placed in -

20°C prior to starting the assay.

First, DNPH (100 µL) was added to each thawed sample, vortexed, and incubated for 10 mins at room temperature. Then, TCA (30 µL) was added to each sample, vortexed, and placed on ice for 5 mins. The samples were then centrifuged at 15000 RPM and the supernatant was discarded. To the remaining pellet, ice cold acetone (500 µL) was added to each tube and the tubes were agitated on the homogenizer for 2 mins at 25 Hz. Once the pellets were visibly dislodged, the samples were incubated for 5 mins at -20°C and centrifuged for 2 mins at 15000

RPM. The supernatant was carefully discarded so as to not disturb the remaining pellet. The acetone wash step was repeated to remove all free DNPH as it can interfere with the final absorbance reading. Finally, 6M guanidine (200µL) was added and the sample was heated in a water bath at 65°C to solubilize the remaining pellet. Following plating, air bubbles were removed, and sample absorbances were read via spectrophotometry in a 96 well plate at an optical density of 375 nm.

33

3.3.4 BCA Protein Assay

As recommended by the ab126287 kit, the Pierce BCA Protein Assay kit (23225,

ThermoFischer Scientific; Waltham, MA, USA) was used to quantify the amount of protein per sample remaining after the carbonylation assay. The kit was stored at room temperature. 6M guanidine was used as the diluent of the samples from the carbonylation reaction, but at this concentration, guanidine interferes with the BCA assay. Samples were diluted with

RNAse/DNAse-free water by a factor of 10 to eliminate interference as well as to fall within the working range of the assay. Thus, 6M guanidine was diluted by a factor of 10 to a final concentration of 0.6M guanidine to prepare the standards as outlined in the microplate protocol.

The working reagent was prepared fresh at a ratio of 50:1 parts of reagent A:B. Each standard or sample (25µL) was loaded onto the microplate followed by the working reagent (200 µL) which was added to each well. The plate was shaken on a plate shaker for 30 seconds and incubated at

37°C for 30 mins. Following incubation, the plate was allowed to return to room temperature for

30 mins, air bubbles in the wells were removed, and the absorbance was read at an optical density of 562 nm. The samples and standard curve values were normalized to a blank of 0.6M guanidine.

3.3.5 Calculations

The carbonyl content was calculated as outlined in the protocol for Ab126287 (Abcam;

Cambridge, GBR).

34 3.4 Glutathione Assay

3.4.1 Tissue prep for glutathione

5% metaphosphoric acid (MPA) was purchased (Sigma-Aldrich; St. Louis, MO, USA) and stored at room temperature. 5% MPA (w/v) was prepared fresh using 10 mL distilled H2O with 0.5 g MPA, vortex mixing, and placed to chill at 4°C. Powdered tissue samples (0.06 g) were homogenized in 5% MPA (1.2 mL) using a chilled steel ball for 2 mins at 25 Hz. The homogenate was centrifuged at 14000 g for 12 mins at 4°C. The supernatant was collected and stored at -80°C for further use.

3.4.2 Glutathione assay

The Glutathione (GSSG/GSH) detection kit was purchased (Cat#:ADI-900-160, Enzo

LifeSci; Farmingdale, NY, USA). Upon receipt, the kit was stored at 4°C. Plate 1 quantifies the combined reduced and oxidized glutathione (GSH+GSSG) in the samples whereas Plate 2 quantifies only the oxidized glutathione (GSSG). Subsequent analyses subtract plate 2 from plate 1 to find the ratio of GSSG to GSH. In healthy tissues, 90% of the glutathione in tissue will be in the reduced form (GSH). In oxidatively stressed tissues, the oxidized form of glutathione will accumulate, resulting in an increased ratio of GSSG to GSH.

Liver samples were diluted by a factor of 80, and placenta samples were diluted by a factor of 30 with 1x assay buffer (supplied as 25x assay buffer). Hippocampal samples were not analyzed due to limited availability of sample tissue. For the first plate, the standard was prepared following the serial dilution scheme outlined in the protocol. 50 µL of each sample or standard was used per well. Freshly prepared reaction mix (supplied, 150µL) was added to each

35 well via a multichannel pipettor. Absorbance was measured at 405 nm each minute for 10 mins.

The second plate was prepared by plating the stock GSSG standard (supplied, 50µL) and the samples and incubating with 4-vinylpyridine (1µL) for 1 hr at room temperature. 4-

Vinylpyridine was purchased (Sigma-Aldrich; St. Louis, MO, USA), and stored at -20°C for one month after opening. The standard was then serially diluted with 1x assay buffer, and freshly prepared reaction mix (150µL) was added. The absorbance was measured similarly to the first plate.

3.4.3 Calculations

The oxidized glutathione content and the reduced glutathione content was calculated as outlined in the protocol for ADI-900-160 (Enzo LifeSci; Farmingdale, NY, USA).

3.5 Positive Control

As a positive control, primary astrocyte cultures from postnatal day 14 guinea pigs were exposed to hydrogen peroxide at concentrations of 100 M, 50 M and 25 M for 6 hrs (n=3). We

chose H2O2 doses which have been shown not to induce apoptosis, but which do induce changes in the expression of genes downstream of the antioxidant response element (ARE) (Bell et al.,

2011). The samples were stored in a 1:10 solution of betamercaptoethanol, and RLT buffer, at -

80°C until RNA was extracted. The in vitro work was done by Andrada Naghi. The mRNA expression for the genes of interest was then quantified by quantitative real-time PCR (qRT-

PCR).

36 3.6 Gene Set Enrichment Analysis

To investigate the long-term oxidative outcomes of sGC treatment in utero, we had the opportunity to investigate a different cohort of animals which were bred as part of another study

(Moisiadis et al., 2017). These animals underwent a similar breeding paradigm, but the pregnant dams were administered 3 courses of sGC or equal volume of saline, on days GD40/41,

GD50/51, and GD60/61 (Moisiadis et al., 2017). The pregnant animals were allowed to deliver at term and the offspring were housed until post-natal day (PND) 40. The animals were sacrificed at a basal state of stress.

The RNA of the hippocampi from female PND40 guinea pigs were sequenced by the

Donnelley Sequencing Centre (Toronto, ON, Canada) and were analyzed for Gene Set

Enrichment Analysis (GSEA). Only female offspring were available to be sequenced at that time. Gene sets were selected from genemania.org based on the keyword ‘oxidative stress’. The top 15 gene sets containing greater than 15 genes were selected for analysis.

3.7 Statistical analyses

Statistical analyses were done in Prism 6.0. Student’s t-tests were used to analyze qRT-

PCR data, where statistical significance was set to p<0.05. The Shapiro-Wilk test was used to evaluate whether or not the data was normally distributed. If not normally distributed, the data was log-transformed and re-analyzed. Outliers were removed based on the appropriate statistical test, ROUT (Q=1%). Data sets were analyzed for homogeneity of variance based on the F-test.

Data sets were log-transformed if the variance was unequal between groups. Similar analyses were done for glutathione ratio. For protein carbonylation, a one-tailed student’s t-test was used.

37 Protein carbonylation is an irreversible post-translational modification (Fedorova et al., 2013).

Therefore if sGC does induce protein carbonylation, we would expect it to only increase following treatment. Thus, a one-tailed t-test is an appropriate statistical test and significance was set accordingly at p<0.1.

Table 3.1. Primer sequences used for qRT-PCR

Gene Name Gene Symbol Sequence Efficiency Catalase Cat Forward: CCT GTG AAC TGT CCC TAC CG 95.5% Reverse: CTT GGT TGT CAG TCA CGC AC Glutathione Peroxidase- GPx4 Forward: ATG TTC AGC AAG GTC TGC GT 98.2% 4 Reverse: TTC CCC TTG GGC TGA ACT TT Glutathione Peroxidase- GPx7 Forward: TCG GTG TCC CTG GTA GTG AA 91.8% 7 Reverse: TCA ATC TCC CTG TCG CTG TC Glyceraldehyde 3- Gapdh Forward: TGT ACT GGA GGT CAA TGA AGG 92.7% phosphate Reverse: GTC GGA GTG AAC GGA TTT G dehydrogenase Hypoxanthine-guanine Hprt1 Forward: AGG CTT GCT CGA GAT GTG AT 106.4% phosphoribosyltranferase Reverse: TCC AGC AGG TCA GCA AAG AAT Peroxiredoxin-3 Prdx3 Forward: TGC AGT GTC AGT GGA TTC CC 102.3% Reverse: CCT TCC AAC AGC ACA CCG TA Peroxiredoxin-6 Prdx6 Forward: GAC AGC TCG CGT GGT ATT CA 96.8% Reverse: TCC TTC CAA TCC ACA GGG GT Superoxide Dismutase-1 Sod1 Forward: TGG GCA AGG GTG GAA ATG AA 98.5% Reverse: AGC ACA CAG CAG GGA ATG TT Superoxide Dismutase-2 Sod2 Forward: CTG AGC CCT AAT GGT GGT GG 97.8% Reverse: TGC AAG CAT CCT CGT TCC TT Thioredoxin Txn Forward: AGG ATG TCG CTG CAG AAT GT 94.1% Reverse: AGC GCC AGA AAA CTC ACT CA Tyrosine 3- Ywhaz Forward: TGG CCC ATC ATG ACA TTG GG 110% Monooxygenase Reverse: GCA CAT GGC CAC CAA ATA GG

38 Table 3.2 Stability of Housekeeping Genes

Housekeeping Gene Stability (p-values) Gapdh Ywhaz Hprt1 GeoMean Female 0.47 0.94 N/A 0.67 Fetal Hippocampus Male 0.29 0.98 N/A 0.57 Female 0.39 0.78 N/A 0.73 Fetal Placenta Male 0.30 0.90 N/A 0.55 Female 0.60 0.49 0.60 0.69 Fetal Liver Male 0.58 0.33 0.62 0.48 Maternal Hippocampus 0.61 0.75 N/A 0.67 PND14 Astrocytes 0.78 0.74 N/A 0.76

Table 3.2. Student’s t-test performed between control treated and sGC treated groups. P-values listed; significance set at p<0.05. There are no significant differences in housekeeping genes: Gapdh, Ywhaz, Hprt1, or the geometric mean between control and betamethasone treated groups in any of the tissues investigated. Hprt1 was used as a housekeeping gene only for fetal liver. The rest of the tissues investigated used only Gapdh and Ywhaz; thus, the corresponding boxes have no value (N/A) for Hprt1.

39 4. Results

4.1.0 Fetal Hippocampus

Student’s t-test analysis indicated that there was no significant differences in gene expression of the antioxidant enzyme genes in the male or female fetal hippocampus following synthetic glucocorticoid (sGC) treatment (p>0.05; Figure 4.1.0A). In the hippocampus, only mRNA studies were carried out due to limitations in sample availability.

4.2.0 Fetal Placenta

Analysis by student’s two-tailed t-test revealed no significant differences in antioxidant enzyme gene expression in male or female placenta between sGC treated and saline treated fetuses ((p>0.05; Figure 4.2.1 A,B).

The protein carbonylation data were analysed using a one-tailed student’s t-test, and significance was set at p < 0.1. Protein carbonylation is an irreversible post-translational modification (Fedorova et al., 2013). Therefore if sGC does induce protein carbonylation, we would expect it to only increase following treatment. Thus, a one-tailed t-test is an appropriate statistical test and significance was set accordingly at p<0.1. In the female or male placenta, there were no significant effects of antenatal sGC treatment on protein carbonylation (p>0.1;

Figure 4.2.2A).

40 The glutathione assay was analyzed using a two-tailed student’s t-test, where significance was set at p<0.05. In the female or male placenta, there was no significant effects of antenatal betamethasone treatment on the total glutathione content, comprising of oxidized and reduced glutathione (p>0.05; Figure 4.2.3A). It was not possible to quantify the ratio of oxidized to reduced glutathione in the fetal placenta using this assay, as the values for oxidized glutathione in the placenta were beyond the limit of detection of the kit.

4.3.0 Fetal Liver

In the fetal male liver, there was a decrease in the expression of peroxiredoxin-6 (Prdx6) associated with prenatal sGC exposure (p<0.05; Figure 4.3.1 A,B). There were no other significant differences in expression of antioxidant enzyme genes in the fetal male or female liver between sGC and control fetuses (p>0.05; Figure 4.3.1 A, B).

There were no significant differences in protein carbonylation in either the fetal female or male liver following antenatal betamethasone treatment (p>0.1; Figure 4.3.2 A).

There were no significant differences in total glutathione content in the male or female liver after sGC exposure (p>0.05; Figure 4.3.3. A). Additionally, there were no significant differences in oxidized to reduced glutathione content in the male or female liver between the betamethasone treated group and the saline treated group (p>0.05; Figure 4.3.3. B).

41 4.4.0 Maternal Hippocampus

Following a two-tailed student’s t-test, there was no significant differences in gene expression of the antioxidant enzymes in the maternal hippocampus following sGC treatement

(p>0.05; Figure 4-4).

4.5.0 Comparative Gene Expression

When comparing the expression of the different antioxidant enzyme genes in the fetal female and male hippocampus, placenta, and liver, two-way ANOVA shows that there was no significant effect of sex on the expression of the genes; thus, both male and female samples were analyzed together, taking the average of littermates to account for litter effects. There was, however, a strong effect of tissue type (p<0.01; Figure 4.5.0A). The data is represented as quantitation cycle number as there was variability in all housekeeping genes between tissues

Highest expression of SOD1, PRDX3, and CAT was observed in the liver, as represented by lower CQ values. Highest expression of TXN, and GPx7 was observed in the placenta. Highest expression of SOD2, and GPX4 was found in the hippocampus. The hippocampus and the liver expressed similar levels of SOD1, TXN, and GPX7, and Prdx6.

4.6.0 Astrocyte Gene Expression (Positive Control)

As a positive control for the genes investigated, astrocytes from post-natal day 14 guinea

pig offspring were cultured and exposed to 100, 50, and 25µM H2O2 or control treatment for 6 hours (n=3). The in vitro work was done by Andrada Naghi. There was significant

42 upregulation of Prdx6 following exposure to all doses of H2O2 compared to the control treated group (p=0.006). Other genes including CAT, TXN, SOD 1, and SOD2 seem to display a trend towards a similar pattern, but did not reach statistical significance.

Appendix : Gene Set Enrichment Analysis of PND40 female hippocampus

In a different study undertaken by Dr. Vasilis Moisiadis, a separate cohort of animals were bred and RNA sequencing was performed on hippocampi of post-natal day 40 female offspring (Moisiadis et al., 2017). These animals were exposed to three courses of betamethasone in utero on gestational days (GD) 40/41, 50/51, and 60/61 as previously described (Iqbal et al., 2012; Moisiadis et al., 2017). To investigate the long-term oxidative stress outcomes of prenatal betamethasone exposure, gene set enrichment analysis (GSEA) was performed. False discovery rate (FDR) significance was set at FDR < 0.25. Normalized enrichment score (NES) significance was set at NES > 1.6. Betamethasone treatment significantly decreased the expression of a set of genes related to oxidative phosphorylation.

43 Figure 4.1.0 - Fetal Hippocampus Gene Expression

4.1.0 A) Gene expression of Catalase (CAT), Peroxiredoxin 6 (Prdx6), Peroxiredoxin 3 (Prdx3) and Glutathione Peroxidase 7 (GPx7) in fetal female and male hippocampus. Expression is relative to the geometric mean of housekeeping genes, GAPDH and YWAHZ. Results shown as ± SEM. Significance set at p<0.05. There was no observed difference between the two treatment groups for any of the target genes.

44

4.1.0 B) Gene expression of Glutathione Peroxidase 4 (GPx4), Thioredoxin (TXN), Superoxide Dismutase 1 (SOD1), and Superoxide Dismutase 2 (SOD2) in fetal female and male hippocampus. Expression is relative to the geometric mean of housekeeping genes, GAPDH and YWAHZ. Results shown as ± SEM. Significance set at p<0.05. There was no observed difference between the two treatment groups for any of the target genes.

45 4.2.1 Fetal Placenta Gene Expression

4.2.1 A) Gene expression of Catalase (CAT), Peroxiredoxin 6 (Prdx6), Peroxiredoxin 3 (Prdx3) and Glutathione Peroxidase 7 (GPx7) in fetal female and male placenta. Expression is relative to the geometric mean of housekeeping genes, GAPDH and YWAHZ. Results shown as ± SEM. Significance set at p<0.05. There was no observed difference between the two treatment groups for any of the target genes.

46

4.2.1 B) Gene expression of Glutathione Peroxidase 4 (GPx4), Thioredoxin (TXN), Superoxide Dismutase 1 (SOD1), and Superoxide Dismutase 2 (SOD2) in fetal female and male placenta. Expression is relative to the geometric mean of housekeeping genes, GAPDH and YWAHZ. Results shown as ± SEM. Significance set at p<0.05. There was no observed difference between the two treatment groups for any of the target genes.

47

4.2.2 Placenta Protein Carbonylation

4.2.2 A) Protein carbonylation in female and male placenta. Values represented are relative to the concentration of protein per sample. Results shown as ± SEM. Significance set at p<0.1. There was no observed difference between the two groups.

4.2.3 Placenta Glutathione (Total)

4.2.3 A) Total glutathione (reduced glutathione (GSH) + oxidized glutathione (GSSG)) in female and male placenta. Values represented are relative to the concentration of sample loaded. Results shown as ± SEM. Significance set at p<0.05. There was no observed difference between the two groups.

48 4.3.1 Fetal Liver Gene Expression

4.3.1 A) Gene expression of Catalase (CAT), Peroxiredoxin 6 (Prdx6), Peroxiredoxin 3 (Prdx3) and Glutathione Peroxidase 7 (GPx7) in fetal female and male liver. Expression is relative to the geometric mean of housekeeping genes, GAPDH,YWAHZ, and HPRT. Results shown as ± SEM. Significance set at p<0.05. Betamethasone treatment significantly decreased the expression of Prdx6 in the male liver.

49

4.3.1 B) Gene expression of Glutathione Peroxidase 4 (GPx4), Thioredoxin (TXN), Superoxide Dismutase 1 (SOD1), and Superoxide Dismutase 2 (SOD2) in fetal female and male liver. Expression is relative to the geometric mean of housekeeping genes, GAPDH, YWAHZ, and HPRT. Results shown as ± SEM. Significance set at p<0.05. There was no observed difference between the two treatment groups for any of the target genes.

50 4.3.2. Liver Protein Carbonylation

4.3.2 A) Protein carbonylation in female and male liver. Values represented are relative to the concentration of protein per sample. Results shown as ± SEM. Significance set at p<0.1. There was no observed difference between the two groups.

51 4.3.3 A - Liver Glutathione (Total)

4.3.3 A) Total glutathione (reduced glutathione (GSH) + oxidized glutathione (GSSG)) in female and male placenta. Values represented are relative to the concentration of sample loaded. Results shown as ± SEM. Significance set at p<0.05. There was no observed difference between the two groups.

4.3.3 B - Liver Glutathione (Ratio)

4.3.3 B). Glutathione ratio (oxidized glutathione (GSSG) : reduced glutathione (GSH)) in female and male liver. Values represented are relative to the concentration of sample loaded. Results shown as ± SEM. Significance set at p<0.05. There was no observed difference between the two groups.

52 4.4.0 Maternal Hippocampus Gene Expression

4.4.0 A) Gene expression of Catalase (CAT), Peroxiredoxin 6 (Prdx6), Peroxiredoxin 3 (Prdx3) and Glutathione Peroxidase 7 (GPx7), Glutathione Peroxidase 4 (GPx4), Thioredoxin (TXN), Superoxide Dismutase 1 (SOD1), and Superoxide Dismutase 2 (SOD2) in maternal hippocampus. Expression is relative to the geometric mean of housekeeping genes, GAPDH and YWAHZ. Results shown as ± SEM. Significance set at p<0.05. There was no observed difference between the two treatment groups for any of the target genes.

53 4.5.0 Comparative Gene Expression

4.5.0 A. Gene expression of Catalase (CAT), Peroxiredoxin 6 (Prdx6), Peroxiredoxin 3 (Prdx3) and Glutathione Peroxidase 7 (GPx7), Glutathione Peroxidase 4 (GPx4), Thioredoxin (TXN), Superoxide Dismutase 1 (SOD1), and Superoxide Dismutase 2 (SOD2) in fetal hippocampus (yellow), placenta (green), and liver (red). Expression displayed as quantitation cycle (CQ) values. Results shown as ± SEM. Highest expression of SOD1, PRDX3, and CAT is observed in the liver. Highest expression of TXN, and GPx7 is observed in the placenta. Highest expression of SOD2, and GPX4 is found in the hippocampus. The hippocampus and the liver express similar levels of SOD1, TXN, and GPX7, and Prdx6.

54 4.6.0 Astrocyte Gene Expression (Positive Control)

4.6.0 A) Gene expression of Catalase (CAT), Peroxiredoxin 6 (Prdx6), Peroxiredoxin 3 (Prdx3) and Glutathione Peroxidase 7 (GPx7), Glutathione Peroxidase 4 (GPx4), Thioredoxin (TXN), Superoxide Dismutase 1 (SOD1), and Superoxide Dismutase 2 (SOD2) in PND14 guinea pig primary culture astrocytes exposed to 100, 50, and 25 µM H2O2 for 6 hours (n=3). Expression is relative to the geometric mean of housekeeping genes, GAPDH and YWAHZ (stable). Results shown as ± SEM. Significance set at p<0.05. There was significant upregulation of Prdx6 following exposure to all doses of H2O2 compared to the control treated group.

55 Appendix 1: Gene Set Enrichment Analysis in Female PND40 Hippocampus

Appendix 1. A) Gene set enrichment analysis on post-natal day 40 (PND40) female hippocampus RNA sequencing data. Gene sets chosen from key work : “Oxidative stress”. False discovery rate (FDR) significance was set at FDR < 0.25. Normalized enrichment score (NES) significance was set at NES > 1.6. Betamethasone treatment decreased the expression of a set of genes related to oxidative phosphorylation.

56 5.0 Discussion

5.1 Hippocampus, Placenta – Gene expression

We observed no difference in gene expression of the key antioxidant enzymes: catalase, thioredoxin, superoxide dismutase, peroxiredoxin, and glutathione peroxidase, between vehicle and sGC treatments, in the placenta or hippocampus in either female or male offspring. These antioxidant enzymes are downstream effectors of the antioxidant response element (ARE) and are the first line of defense against increased free radical production in the cell. An increase in the expression of these enzymes following sGC exposure would be indicative of an increased response to oxidative stress. Increased expression of these genes has been associated with an increased protein expression of these enzymes (Mates, 2000).

These results observed in the hippocampus are contrary to our hypothesis, which could suggest that antioxidant enzymes are not transcriptionally regulated in these tissues following sGC exposure, but rather through post-translational modifications. There is evidence in the literature for peroxiredoxin to be regulated through reversible sulfinylation, and catalase and glutathione peroxidase to be regulated through phosphorylation (Rhee et al., 2005). An increase in the proportion of the active form compared to the inactive form of the enzymes following sGC exposure would suggest an increased need to regulate cellular H2O2, and thus would serve as an indirect measure of increased H2O2. Another measure might be enzymatic activity of the tissue, where an increased enzyme activity in the sGC treated groups, would suggest increased antioxidant response of the tissue.

57

It might be possible that while the control group establishes a baseline expression of the antioxidant enzyme genes, the group treated with sGC is attempting to respond by mounting an antioxidative response through increased expression of genes downstream of the antioxidant response element (ARE). However, this transcriptional response might be inhibited by the presence of active glucocorticoid receptor, which prevents binding of CREB binding protein

(CBP) to the ARE and inhibits transcription (Alam et al., 2017). This would suggest that if sGC induces an increase in the oxidative state of the cell, the treatment is also inhibiting a full protective response, which could further exacerbate the oxidative imbalance of the cell. Thus, while we observe no differences in antioxidant enzyme expression following sGC treatment, we might actually be observing attenuated transcription of the protective ARE genes, which happens to be leveled with the established baseline expression of the control group.

5.2 Liver – Gene expression

In the fetal liver, we observed a significant decrease in gene expression of Prdx6 mRNA levels in males. Our results in the liver are also contrary to our hypothesis. A recent study by

Alam et al in 2017 proposed a mechanism for sGC-induced reduction in antioxidant enzyme expression in the liver. Normal response to oxidative stress includes signaling through nuclear factor erythroid 2-related factor 2 (Nrf2), a transcription factor that can bind the antioxidant response element (ARE) of a gene through association with CREB binding protein (CBP). Alam et al. suggests that following sGC signaling, the presence of activated glucocorticoid receptor prevents Nrf2 association with CBP, inhibiting the transcription of genes downstream of the

ARE, including genes for antioxidant enzymes (Ma et al., 2013; Hayes et al., 2014; Malhotra et

58 al., 2010). This group further showed increased measures of oxidative stress through the sGC- induced downregulation of the antioxidant enzyme genes. Our gene expression data in the liver is in support of this mechanism. However, similar results are not observed in the placenta or hippocampus.

5.3 Placenta, Liver - Protein Carbonylation, Glutathione Ratio

Our results indicate that sGC treatment does not significantly alter protein carbonylation or total glutathione in the placenta, which is consistent with the lack of sGC effects on placental gene expression. Interestingly, there were also no effects of sGC on protein carbonylation or the glutathione ratio in the liver. Given the reduction in antioxidant enzyme gene expression, we would expect to see an increase in oxidative damage. This could suggest that we were capturing a short window, 24 hours, following the last exposure to sGC. It has been shown that markers of oxidative stress including lipid peroxidation as well as glutathione peroxidase activity are elevated following sGC exposure up to 72 hours following last exposure (Verhaeghe et al.,

2009). However, it might be that we are missing the oxidative damage following the second sGC exposure in the 24 hour window that we captured. In an in vitro model, You et al., observed that following sGC exposure, ROS levels peak after 4 hours, and return to baseline after 8 hours (You et al., 2009). It is important to note that previous studies have shown that a single course of prenatal sGC (beta) in rats leads to a significant decrease in proliferation, but not induction of apoptosis (Scheepens et al., 2003). Therefore, it is unlikely that we are not capturing the oxidative damage due to apoptosis.

59 5.4 Maternal Hippocampus – Gene expression

There is evidence to support increased antioxidant enzyme activity in specific brain regions following a high sGC dose (10 mg/kg corticosterone; daily for 3 days) in adult rodents

(McIntosh et al., 1998). However, there is no evidence that sGC at a dose of 1mg/kg induces oxidative stress in the adult hippocampus in a model of the guinea pig. Thus, the maternal hippocampus was investigated for changes in antioxidant enzyme gene expression. Additionally, we wanted to investigate whether sGC induced an antioxidant response in adult hippocampi compared to fetal hippocampi. Gene expression data from maternal hippocampus showed no difference in any antioxidant genes.

Normal pregnancy is associated with changes in oxidative potential. A small study comparing pregnant (n=17) and non-pregnant women (n=12) found that pregnancy was associated with an initial decrease in maternal venous total antioxidant capacity (TAC) in the first trimester that gradually increased over gestation and exceeded control levels up to 8 weeks post-partum (Toescu et al., 2002). We did not observe any significant change in the expression of antioxidant enzyme genes between control treated maternal hippocampus and sGC treated maternal hippocampus. This might be due to the endogenous antioxidant levels increasing during late gestation. Therefore, the sGC induced ROS might be sufficiently inhibited by the autogenous upregulation of antioxidant species, at this time.

5.5 Comparative Gene expression in Tissues

The lack of sGC-induced changes in antioxidant enzyme gene expression in the placenta and hippocampus could suggest that there are different antioxidant mechanisms in these tissues

60 that respond to GC. Another possibility is that the different tissues exhibit different levels of endogenous antioxidant responses which will affect the degree of response to sGC. To investigate this, we randomly selected 6 control-treated samples from each tissue type. These samples were analyzed together for basal antioxidant gene expression. Following a two-way

ANOVA, there was no effect of sex on gene expression, but a strong and consistent effect of tissue type on the gene expression. Thus, subsequent analyses combined male and female data after controlling for litter effects. One-way ANOVA and Tukey’s multiple comparison post-hoc analysis revealed the highest expression of SOD1, PRDX3, and CAT observed in the liver.

Highest expression of TXN, and GPx7 was observed in the placenta. Highest expression of

SOD2, and GPX4 was found in the hippocampus. The hippocampus and the liver expressed similar levels of TXN, and GPX7, and Prdx6.

The liver is a GC responsive organ, and is exposed to a variety of toxins and xenobiotics.

During the perinatal period, the human fetal liver begins to express enzymes required for xenobiotic clearance, and close to term, the fetal liver begins to prepare for gluconeogenesis

(Fowden et al., 2016). Thus, it may require more endogenous antioxidant protective mechanisms compared to other organs, particularly at this gestational time point.

It is known that the brain endogenously exhibits low levels of antioxidant enzymes

(Ostracovitch, 2001). The hippocampus has other protective structures such as the blood-brain barrier, and thus might have less of a need for endogenous antioxidant mechanisms. While these results are based on a pilot study with a small number of samples, it is interesting to see tissue- specificity amongst the different antioxidant enzyme genes at the same time point of GD52.

Further studies need to be undertaken to validate these findings at the level of protein.

61 5.6 Astrocyte exposure to H2O2

We observed that following H2O2 exposure (100, 50, and 25µM for 6 hours), selected

ARE target genes did show changes in expression following the oxidative challenge.

Particularly, Prdx6 treated with all three doses of H2O2 was significantly upregulated compared to control treated astrocytes. Additionally, CAT, TXN, SOD1, and SOD2 followed a similar pattern, however statistical significance was not reached, possibly due to the low sample size

(n=3). Therefore, if there were any changes in oxidative stress following sGC treatment, alterations in gene expression in the target genes would be indicative of oxidative challenge.

However, some genes did not show expected increases in expression. This might be because the

dose of H2O2 that we treated with might not be high enough to induce the expression of all of the genes, but only those necessary to defend against preliminary ROS production. These results might also be due to the low sample size (n=3).

5.7 Physiological ROS levels

sGC decreases proliferation (re-starting cell cycle (Ostrakhovitch & Semenikhin, 2013)) and favors differentiation (exiting the cell cycle (Ostrakhovitch & Semenikhin, 2013)) (Fowden et al., 2016). Low ROS signals for proliferation, whereas high ROS signals for differentiation

(Ostrakhovitch & Semenikhin, 2013). While we do not observe changes in sGC-induced changes in Nrf2 genes, this might be due to the fact that sGC signals for differentiation through inducing physiologically appropriate changes in ROS levels. However, since the changes in

ROS levels would be at physiological levels, no changes in antioxidant enzymes should be observed (Ostracovitch & Semenikhin, 2013). Additionally, inducing altered antioxidant responses to developmental increases in ROS could be detrimental and can lead to abnormal

62 development (Lee & Davis, 2011). Therefore, it is possible that while sGC exposure in utero is inducing increases in ROS, levels of ROS are not sufficient to induce a protective effect, but are necessary and sufficient to induce changes in cell cycle progression and tissue development.

5.8 Timing, Mode of delivery

The observed results are contrary to our hypothesis that following antenatal sGC therapy, there would be increased markers of oxidative damage through increased protein carbonylation, increased glutathione ratio, as well as increased expression of antioxidant enzyme genes. This might be due to the time point at which we investigated. Fetal tissues were collected 24 hours following last exposure to sGC. One study investigated the effect of timing of sGC dose and mode of delivery on oxidative stress outcomes. In human umbilical vein blood, malondialdehyde levels remained elevated up to 72 hours following last exposure to sGC and

GPx3 levels were reduced within the first 24 hours following sGC exposure (Verhaeghe et al.,

2009). They did not observe any significant differences in oxidative stress measures based on mode of delivery or gestational age. One important limitation of this study was that the authors included mothers treated with sGC and who subsequently delivered at different time points, but did not include a non-treated group. Thus, if they conducted a separate study in an animal model comparing a 24 hour group to a non-treated group, they might not have seen any significant differences. Additionally, in the present study, we observed a decrease in Prdx6 levels in male livers. Perhaps we are beginning to see changes in oxidative potential at this time point, and if we further investigate the animals 72 hours following the last exposure, we might see further increases in oxidative stress. Therefore, these results demonstrate that the timing of delivery following sGC exposure has a significant impact on oxidative stress outcomes.

63

This discrepancy could also point towards the differences in oxidative capacity in fetal compared to neonatal environments. During the perinatal period, the fetus undergoes many changes in oxidative status. The fetus increases the production of antioxidant enzyme throughout the last 15% of gestation in preparation for neonatal life (Davis & Auten, 2010).

While Verhaeghe et al. did not find oxidative damage following sGC therapy to be correlated with mode of delivery or gestational age, other studies have shown both of these parameters to be large factors to the preparation of the fetus for the neonatal environment. Labor is an oxidatively challenging event, and infants delivered via cesarean section might have lower oxidative burden, and thus lower antioxidant enzyme expression as well as activity (Davis &

Auten, 2010; Georgeson et al., 2002). Indeed, neonates of cesarean delivery without labor had reduced levels of antioxidant enzymes in both term and preterm cases when compared to infants delivered vaginally (Georgeson et al., 2002). Gestational age is also an important factor as

Georgeson et al. observed preterm infants to have a significantly lower antioxidant potential when compared to term infants (Georgeson et al., 2002). The authors did not specify sGC exposure in the samples, but these studies suggest that further research needs to be done to explore the effect of timing of glucocorticoid exposure, mode of delivery, and gestational age on the oxidative stress outcomes of sGC treated offspring.

5.9 Nutrition

Ascorbic acid (Vit C) is a major nutritional antioxidant, along with vitamins A and E. Vit

C in particular helps scavenge for the hydroxyl radical as well as lipid hydroperoxides and supplementation of these antioxidants have been studied in the context of preeclampsia and normal birth (Lee & Davis, 2011). One randomized controlled study showed that Vit C and/or

64 Vit E supplementation does not reduce the risk of gestational hypertension or preeclampsia

(Villar et al., 2009). However, another small study showed that Vit C supplementation during pregnancy reduced malondialdehyde levels when compared to a non-supplemented pregnant group (Olayaki et al., 2008). Therefore, the role of Vit C supplementation during pregnancy is not yet well understood.

The guinea pig diet is supplemented with ascorbic acid (Vit C) as they are unable to synthesize it, similar to humans and other primates (Canadian Council on Animal Care). Adults are supplemented with up to 10 mg/kg ascorbic acid, whereas during pregnancy, 30 mg/kg may be required for optimal gestational conditions (Canadian Council on Animal Care). In guinea pigs, Vit C supplementation prevents immune deficiencies and reduces the risk of developing scurvy. However, in the chow, Vit C is quite unstable and can degrade if stored at non-optimal conditions. Since this is a variable that was not able to be fully controlled, the Vit C supplementation in guinea pigs during their gestation might have had a part in masking some oxidative stress outcomes that might follow sGC therapy.

5.10 No Acute Effect of sGC in Fetus

While we do observe the male liver to downregulate Prdx6 gene expression following sGC treatment, our other measures of oxidative stress in the fetus could suggest that the fetus is largely protected from acute oxidative stress following sGC exposure. We did not observe any changes in protein carbonylation or glutathione ratio, which suggests that 24 hours following a second sGC exposure, there is no oxidative damage. Future studies should investigate lipid peroxidation and DNA peroxidation to further validate these findings.

65 5.11 Long-term outcomes : PND40 Female Hippocampus – GSEA

In this study, we demonstrate that the fetus is largely protected from acute oxidative stress following sGC treatment. However, mounting evidence suggests that there are several oxidative long-term consequences of GC exposure in utero (de Vries et al., 2007; Haussmann et al., 2012; Marasco et al., 2013). We have begun to investigate this possibility by performing gene set enrichment analysis (GSEA) on RNA sequenced from post-natal day (PND) 40 female hippocampi, that were last exposed to sGC on GD60 and delivered at term. In female PND40 offspring, we found that sGC treatment decreased expression of a set of genes related to oxidative phosphorylation, a key mechanism of the mitochondria (See Appendix 1). Since the mitochondria is the site of the majority of ROS production, this might suggest that following antenatal exposure to sGC, mitochondrial function and subsequent baseline ROS balance is being altered later on in the life of the offspring. Although the PND 40 animals were subjected to a three courses of sGC in utero as opposed to the two course paradigm in animals investigated in the current study, the available tissue and data allowed us to try and begin to make comparisons between fetal exposure to sGC and oxidative stress, later in life. These results might point towards the mitochondria as a focus for future studies in this context.

Our results thus far suggest that there might be long-term programming of mitochondrial functions following glucocorticoid exposure in utero. We found that sGC significantly reduced the expression of a set of genes related to oxidative phosphorylation, 50 days following last exposure. There is precedence for GC reducing mitochondrial activity. In 2013, Tang et al found corticosterone administration in rat pheochromocytoma cells (adrenal medulla) at 0.031,

0.063,and 0.125 mmol/L for 24 hrs to decrease the activity of the mitochondrial complex 1, but

66 not of complex 3 (Tang et al., 2013). Interestingly, this decrease in mitochondrial activity was associated with increased protein carbonylation, whereas a reduction in mitochondrial activity would be expected to result in a reduction of oxidative stress, since the mitochondria is a major site of ROS production (Tang et al., 2013). Another study investigated the effect of chronic restraint stress on oxidative stress in rat hippocampi (Madrigal et al., 2001). They observed that following 21 days of daily restraint stress, that the activity of the first mitochondrial complex was inhibited, but complex 4 was not affected (Madrigal et al., 2001).

Some studies have found that following sGC exposure, activated glucocorticoid receptors can increase the transcriptional activity of the tumor suppressor protein, p53 (Crochemore et al.,

2002). Under low stress conditions, small increases in p53 activity have been associated with decreased ROS production, cell cycle arrest, and lower mitochondrial activity (Vousden et al.,

2009). One study, observed downstream targets of p53 to be activated following endogenous and exogenous oxidative stress in a rodent model of SOD2-/- and GPx-/- (Han et al., 2008). p53 has also been observed to be an endogenous inhibitor of Nrf2 transcription factor (Namani et al.,

2014; Faraonio et al., 2006). We have shown decreased gene set enrichment related to oxidative phosphorylation following sGC treatment in utero in post-natal day 40 animals. This might be due to activated glucocorticoid receptors mediating changes in mitochondrial activity through p53. In this case, a small increase in p53 could be inducing a protective environment for the cell, through altering the state of the cell cycle via ROS levels. In addition, drugs affecting development, including sGC, have been associated with decreases in cellular proliferation and increases in differentiation (Carson et al., 2016). However, further studies need to be done to understand how p53 affects ROS in this mechanism.

67 5.12 Limitations

A key limitation of this study is that all tissues were collected and snap-frozen prior to analysis. The nature of redox species makes it very reactive and particularly sensitive to handling procedures. Many widely-used assays for total redox content of a tissue depends on fresh, unfrozen tissue, treated at the time of collection. As this is was not possible for our study, we used indirect measures of oxidative stress. However, since both groups are handled equally, we hope to negate any of these effects. In the literature, other measures include urinary 8-

OHdG, as well as mitochondrial damage and antioxidant enzyme activity. However, reliable measurements of antioxidant enzyme activity cannot be made on frozen tissue (Bilodeau et al.,

2000).

Another limitation of this study was that samples were collected as part of a larger study.

While this allowed for a greater variety of questions to be asked and conclusions to be made, the sample quantity was greatly limited per animal. The hippocampal size was particularly small, and thus a major arm of this study was not able to be fully explored through protein carbonylation and glutathione ratio.

Within the tissues that were available, prior to analysis, the different cell types were not isolated. It has been shown that different cells have different endogenous antioxidant capacities

(Bell et al., 2011). This could have significantly masked changes in the oxidative stress outcome measures in more oxidatively reactive cell types such as astrocytes compared to neurons in the hippocampus (Bell et al., 2011). Since the data presented in this project is based on homogenized tissue, we are limited in drawing fine-tuned conclusions. Future directions

68 should focus on differentiating different cell types in the hippocampus, placenta, and liver.

Future studies should also isolate target organelles such as mitochondria and nuclei to investigate the cellular localization of proteins such as p53.

A fourth limitation of this study was that the guinea pig genome is not as well annotated as human or rodent species (Carter et al., 2007). Therefore, some genes that were downstream of the antioxidant response element (ARE) were not able to be investigated. Moreover, while we were fortunate to be able to begin to investigate the long-term oxidative outcomes of sGC treatment in utero, the long-term cohort was treated with three courses of sGC in utero, whereas the animals investigated at GD52 were treated with two courses of sGC in utero. Therefore, these two study designs differed in the number of exposures to sGC, and thus direct comparisons cannot be drawn.

5.13 Future directions

We have shown preliminary data that the hippocampal mitochondria might undergo long- term programming following sGC exposure in utero. Future studies might isolate mitochondria from different tissues including the hippocampus and the liver to investigate mitochondrial complex activities as well as oxidative phosphorylation activity in fetal and juvenile offspring.

Some studies have shown elevated GC exposure to negatively impact mitochondrial activity

(Madrigal et al., 2001; Tang et al., 2013). However, no study has investigated this in a gestational model. Moreover, no study has investigated the ontogeny of mitochondrial dysfunction following sGC exposure in utero.

69

In the field of developmental origins of health and disease (DOHAD), there has been mounting evidence in support of the glucocorticoid-induced vulnerability hypothesis. This concept suggests early life exposure to stress predisposes offspring to developing cardiac, metabolic, and endocrine dysfunction later in life following a secondary stressor, possibly due to reprogramming of the hypothalamus-pituitary-adrenal (HPA) axis (Conrad, 2008). Ahlbom et al., in 2011 observed this phenomena in an in vitro model where they cultured cerebellar granule cells from week-old rats exposed to sGC in utero. They then exposed these cells to hydrogen peroxide and showed that following a secondary oxidative stressor, these cells had significantly lower catalase activity (Ahlbom et al., 2011). Thus, future directions could investigate this in an in vivo gestational model of GC exposure. One study might compare juvenile offspring treated with two courses of glucocorticoids and another group who undergo a secondary nutritional, behavioural, or physical stressor. We would expect to see an increase in oxidative damage markers in tissues and plasma collected from the second, stressed group.

P53 might play a role in modulating the oxidative changes following sGC treatment. One study to investigate this could be to quantify the expression of p53 in tissues following sGC therapy. P53 is localized to the nucleus following activation, thus specifying the different cellular localization is important (Han et al., 2008). Through differential centrifugation, different organelles could be isolated and western blots could be run to quantify levels of p53 protein in both fetal and juvenile offspring of sGC therapy compared to offspring of saline treated animals.

Follow up studies could include measuring which state of the cell cycle the cells are in, and additionally, if there are any changes in the ROS content or mitochondrial activity of the tissues.

70

One hypothesis as to why we are not seeing any changes in antioxidant enzyme expression, is that the sGC are leading to increased ROS, but only to physiological levels, which might induce changes in cell cycle to favor differentiation over proliferation. The sGC might not be inducing pathological levels of ROS which would then trigger increased expression of antioxidant enzymes as a protective response. One study to begin to investigate this hypothesis might be to treat an in vitro culture with hydrogen peroxide at increasing levels to get a gradient of ROS measures from those which induce antioxidant enzyme expression, to apoptosis. Then, this could act as a standard curve to which ROS levels are compared from in vivo subjects.

Future studies should explore additional markers of lipid peroxidation in these tissues.

We were unable to quantify lipid peroxidation through the FOX2 assay on lipid extracts since the levels in the tissue samples were beyond the limit of detection for the kit. Other studies investigate lipid peroxidation through quantifying malondialdehyde levels through the TBARS assay, or comparing F2-isoprostanes through mass spectroscopy (Madrigal et al., 2001; Zhu et al., 2008). Another, less common measure of lipid peroxidation would be to measure proteins tagged with 4-HNE through a western blot (Andringa et al., 2014).

5.14 Significance and Conclusions

In this study, we demonstrate that sGC does not have an acute effect on antioxidant enzymes at the level of transcription or oxidative damage in the hippocampus or the placenta, but the fetal liver might be more susceptible. We also investigated the acute effect of sGC on fetal

71 levels of protein carbonylation and the glutathione ratio in the placenta and liver. These results reinforced the data from the transcriptional analysis, whereby there was no change in the fetal hippocampus or placenta, and there were also no changes observed in the fetal liver.

We have also begun to investigate long-term oxidative outcomes of sGC therapy. We have shown that sGC treatment in utero might induce long-term programming of oxidative phosphorylation in the hippocampi through gene set enrichment analysis. Future studies should consider the role of mitochondria in this context.

This study advances the understanding of the short-term and long-term consequences of antenatal sGC therapy and the mechanisms that underlie them. These data will help uncover the potential role of oxidative stress as a mechanism of sGC related effects. This study is clinically relevant as up to 10% of births are at risk of preterm delivery and most these women will be treated with sGC therapy (Moisiadis et al., 2014).

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