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The effects of low molar concentrations of bisphenol A on neuron viability and survival under neurotoxic… Tong, Jade Pui Wai 2010

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THE EFFECTS OF LOW MOLAR CONCENTRATIONS OF BISPHENOL A ON NEURON VIABILITY AND SURVIVAL UNDER NEUROTOXIC STRESS by Jade Pui Wai Tong B.Sc., The University of British Columbia 2008  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  MASTER OF SCIENCE  in  The Faculty of Graduate Studies (Neuroscience) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) October 2010 c Jade Pui Wai Tong, 2010  Abstract Bisphenol A (BPA) is an ubiquitous environmental xenoestrogen excreted in the urine of 95-99% of humans studied. In this investigation, we examine the effects of 1mM to 10fM concentrations of BPA on the viability of rat cortical neurons under acute (5h) and chronic (DIV3-9, DIV3-15) exposure conditions. Post-exposure, we also challenged the cultures with an oxidative or excitotoxic stress to determine whether BPA conferred neuroprotection, susceptibility or neither to the challenged cultures. Finally, we studied the effects of different concentrations of BPA on the activation of SREBP 1, a transcription factor that regulates many genes with important effects on neuron function. We found that 1mM and 500uM BPA are neurotoxic under chronic exposure, but only 1mM BPA decreased cell viability after 5h. Acute and chronic exposure to BPA conferred neither neuroprotection nor susceptibility to oxidative or excitotoxic stress in the neuron cultures. Five hours exposure to 10pM and 1pM BPA increased SREBP 1 activation two-fold. In DIV9 cultures, 10pM BPA stimulated a maximal three-fold SREBP 1 activation at 8h post exposure, while at DIV15, 10pM BPA stimulated a maximal two-fold SREBP 1 activation at 5h post exposure. High, physiologically irrelevant concentrations of BPA induce neuronal cell death, and while sublethal concentrations do not predispose cultures to oxidative or excitotoxic stress, they also do not confer neuroprotection as a true estrogen, estradiol, would. Sublethal concentrations of BPA activate SREBP 1 in a hormetic manner, resulting in a non-monotonic dose response curve.  ii  Table of Contents Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  ii  Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  vii  Dedication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . viii Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  ix  1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  1  1.1  Bisphenol A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  1  1.1.1  Structure and Biochemical Properties . . . . . . . . . . . . . .  1  1.1.2  Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . .  1  1.1.3  Industrial Use . . . . . . . . . . . . . . . . . . . . . . . . . . .  2  1.1.4  Environmental Relevance . . . . . . . . . . . . . . . . . . . . .  3  1.1.5  Human Exposure to BPA . . . . . . . . . . . . . . . . . . . .  4  1.1.6  In Vitro Studies . . . . . . . . . . . . . . . . . . . . . . . . . .  5  1.1.7  In Vivo Studies . . . . . . . . . . . . . . . . . . . . . . . . . .  6  1.1.8  Non-Monotonic Dose Response . . . . . . . . . . . . . . . . .  8  1.1.9  Controversies . . . . . . . . . . . . . . . . . . . . . . . . . . .  9  Sterol Regulatory Element Binding Protein 1 . . . . . . . . . . . . . .  11  1.2.1  Functions of SREBP 1 . . . . . . . . . . . . . . . . . . . . . .  11  1.2.2  Model System . . . . . . . . . . . . . . . . . . . . . . . . . . .  13  Aims of Thesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  15  2 Effects of Acute and Chronic BPA Exposure on Neuronal Health  20  1.2  1.3  2.1  Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  20  2.1.1  20  MTT Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . .  iii  2.1.2  Aims of Chapter . . . . . . . . . . . . . . . . . . . . . . . . .  21  Materials and Methods . . . . . . . . . . . . . . . . . . . . . . . . . .  22  2.2.1  Primary Rat Neuron Culture and Maintenance . . . . . . . . .  22  2.2.2  BPA Treatment . . . . . . . . . . . . . . . . . . . . . . . . . .  23  2.2.3  MTT Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . .  23  2.2.4  Statistical Analysis . . . . . . . . . . . . . . . . . . . . . . . .  23  2.3  Study Design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  24  2.4  Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  25  2.4.1  Effect of Acute BPA Exposure on Cortical Neuron Health . .  25  2.4.2  Effect of Chronic BPA Exposure on Cortical Neuron Health .  25  Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  27  2.2  2.5  3 Effects of BPA on Neuron Response to Excitotoxic and Oxidative Stress Challenge . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  34  3.1  Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  34  3.1.1  Neurotoxic Stress Challenge . . . . . . . . . . . . . . . . . . .  35  3.1.2  Aims of Chapter . . . . . . . . . . . . . . . . . . . . . . . . .  36  Materials and Methods . . . . . . . . . . . . . . . . . . . . . . . . . .  37  3.2.1  Primary Rat Neuron Culture and Maintenance . . . . . . . . .  37  3.2.2  BPA Treatment . . . . . . . . . . . . . . . . . . . . . . . . . .  37  3.2.3  Oxidative and Excitotoxic Stress Challenge . . . . . . . . . . .  37  3.2.4  MTT Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . .  38  3.2.5  Statistical Analysis . . . . . . . . . . . . . . . . . . . . . . . .  38  3.3  Study Design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  39  3.4  Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  41  3.2  3.4.1  Effect of Acute BPA Exposure on Neuron Response to Excitotoxic and Oxidative Stress Challenge . . . . . . . . . . . . . .  3.4.2  3.5  41  Effect of Chronic BPA Exposure on Neuron Response to Excitotoxic and Oxidative Stress Challenge . . . . . . . . . . . . .  41  Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  43  4 Effects of BPA on Cell Regulation Mechanisms in Cortical Neurons 51  iv  4.1  Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  51  4.1.1  Sterol Regulatory Element Binding Protein 1 . . . . . . . . . .  52  4.1.2  Aims of Chapter . . . . . . . . . . . . . . . . . . . . . . . . .  52  Materials and Methods . . . . . . . . . . . . . . . . . . . . . . . . . .  53  4.2.1  Primary Rat Neuron Culture and Maintenance . . . . . . . . .  53  4.2.2  BPA Treatment . . . . . . . . . . . . . . . . . . . . . . . . . .  53  4.2.3  Sample Preparation . . . . . . . . . . . . . . . . . . . . . . . .  53  4.2.4  Protein Quantification . . . . . . . . . . . . . . . . . . . . . .  54  4.2.5  Western Blotting . . . . . . . . . . . . . . . . . . . . . . . . .  54  4.2.6  Statistical Analysis . . . . . . . . . . . . . . . . . . . . . . . .  55  4.3  Study Design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  56  4.4  Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  57  4.2  4.4.1  Effect of Various Concentrations of BPA on Activated SREBP 1 after 5h . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  57  Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  58  5 Conclusions and Further Studies . . . . . . . . . . . . . . . . . . . .  60  Bibliography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  65  4.5  v  List of Figures 1.1  Structure of Bisphenol A. . . . . . . . . . . . . . . . . . . . . . . . .  16  1.2  Structure of 17β-estradiol. . . . . . . . . . . . . . . . . . . . . . . . .  17  1.3  Structure of genistein. . . . . . . . . . . . . . . . . . . . . . . . . . .  18  1.4  SREBP 1 activation as a transcription factor through cleavage by S1P and S2P . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  19  2.1  Effect of 5h acute BPA treatment on the viability of DIV15 neurons.  29  2.2  Effect of acute 10pM BPA treatment on the viability of DIV9 neurons.  30  2.3  Effect of acute 10pM BPA treatment on the viability of DIV15 neurons. 31  2.4  Effect of chronic BPA exposure on the viability of DIV9 neurons. . .  32  2.5  Effect of chronic BPA exposure on the viability of DIV15 neurons. . .  33  3.1  Effect of acute BPA pre-treatment on DIV9 cortical neuron response to excitotoxic and oxidative stress. . . . . . . . . . . . . . . . . . . .  3.2  Effect of acute BPA pre-treatment on DIV15 cortical neuron response to excitotoxic and oxidative stress. . . . . . . . . . . . . . . . . . . .  3.3  48  Effect of chronic BPA pre-treatment on DIV9 cortical neuron response to excitotoxic and oxidative stress. . . . . . . . . . . . . . . . . . . .  3.4  47  49  Effect of chronic BPA pre-treatment on DIV15 cortical neuron response to excitotoxic and oxidative stress. . . . . . . . . . . . . . . . . . . .  50  4.1  Effect of 5h BPA treatment on SREBP 1 expression. . . . . . . . . .  59  4.2  Quantified effect of 5h BPA treatment on SREBP 1 expression. . . .  59  vi  Acknowledgements I would like to thank my supervisor, Dr. Max Cynader, for his help, suggestions and guidance throughout my graduate studies. Without his encouragement to explore and expand my knowledge of the neurosciences at the beginning of my program, I would not have started an investigation that eventually linked molecular and cellular neuroscience with toxicology. This thesis would not have been possible without the help of Dr. Changiz Taghibiglou, whom I would like to thank firstly, for steering me towards the role of BPA in activating SREBP 1, secondly, for supplying me with his SREBP 1 antibody and finally, for being a valuable mentor in encouraging and guiding me throughout my troubleshooting. I am also grateful to my committee members Dr. Stelvio Bandiera, Dr. Liisa Galea and Dr. Ann Marie Craig for their guidance and help in formulating my experimental questions and their suggestions to the organization of my thesis. I am indebted to Wendy Wen for her patience in teaching me and acquiescing to the demands of my many early ambitious experiments that never made it into this thesis. Last, but definitely not least, I am thankful to my friends Kaiyun Yang, Colette Chiu, Eddie Pokrishevsky, Melanie Bertrand and Shanshan Zhu for their support and encouragement during the worst of times, and their good cheer and company during the best of times.  vii  To my mother, Ann Yu, who does not understand why I choose the paths I do but accepts them regardless for the sake of my happiness, and my siblings, Derek and Jackie Tong, for their unconditional support and love.  viii  Preface Animal Ethics Certificate Number: 3152-08 Practical Animal Care Training Certificate Number: RBH-621-09 Timed Pregnant Rats Protocol Number: A10-0169  ix  Chapter 1 Introduction 1.1 1.1.1  Bisphenol A Structure and Biochemical Properties  Bisphenol A (BPA) is an organic compound composed of two phenol rings (Figure 1.1). It is primarily this phenolic group, which is also present in the naturally occuring endogenous estrogen 17β-estradiol and the phytoestrogen genistein, that gives BPA the ability to bind to estrogen receptors α and β (Figures 1.2 and 1.3) [1, 2, 3, 4, 5, 6, 7]. In addition to the well-known estrogen receptors α and β, BPA is also able to bind to a membrane-bound and a transmembrane G protein-coupled estrogen receptor as well as an orphan nuclear receptor and aryl hydrocarbon receptor [8, 9, 10, 11]. As a result, BPA has the potential to mimic a variety of estrogen functions in the cell [1].  1.1.2  Metabolism  BPA is glucuronidated by UDP-glucuronosyltransferase (UGT) liver enzymes and excreted in urine [12, 13, 14]. Unglucuronidated BPA is excreted in feces [12, 13, 15, 16]. Volkel et al (2002) were only able to detect glucuronidated BPA as a metabolite at physiologically relevant levels of exposure, however, the detection limits in their study were in the nanomolar range, which may not be sufficient to detect the results of BPA metabolism via a different pathway [14]. Glucuronidated BPA has no estrogenic  1  activity, unlike free BPA [14]. A previous study found the complete elimination of labeled BPA from human subjects occurred within 24h [14]; however, it is important to remember that human exposure to BPA is constant and not a one-time dosage. Only about 1% of all ingested, absorbed or inhaled BPA is estimated to remain in adult tissues [12, 17]. At the same time, it is important to note that UGT liver enzyme activity has not been found in human nor rat fetal livers, and so the percent of BPA retained in the fetal body is likely to be higher than that of the adult [18, 19]. Taking into consideration the sensitivity of the fetal brain to circulating hormones and chemicals, it is not unlikely that BPA has some effect on the development of the fetal brain in both rats and humans [20, 21].  1.1.3  Industrial Use  BPA is an additive used in the production of almost all polycarbonate plastics and epoxies due to its ability to confer endurance to clear plastics [1, 6, 12]. In addition to plastic food containers, BPA is also be found in the lining of metal cans, dental sealants, coatings, finishes, pipes and a variety of household appliances [12, 22]. Over the last decade, the production of BPA has exceeded 6 billion pounds per year [1]. A major concern surrounding this massive global usage of BPA in everyday items is the ability of BPA to leach out of plastics over time and into our food and water supply [1, 23, 24]. Exposure of humans to BPA is primarily by ingestion, with secondary exposure by absorption through skin and inhalation through lungs [12, 23, 25]. BPA is present even in non-plastic materials, such as canned soft drinks, as it is used in the production of the epoxy used to line the insides of the can [1, 24]. A recent wide-ranging study by Health Canada in 2009 found low levels of BPA in every canned soft drink, energy drink and fruit drink tested. BPA is also found in the lining of drinking water storage tanks and wine vats and migration under normal storage conditions has been measured [26, 14]. While the rate of BPA migration from plastic to food is primarily determined by the conditions in the manufacturing process, exposure of the plastic to heat or acids can increase the amount of leaching that occurs [12, 27, 28]. As a result, usage of any plastic containing BPA in contact  2  with foods or water will result in some contamination over time.  1.1.4  Environmental Relevance  The actual degree to which BPA has permeated our environment is hard to determine because it is difficult to remove all background contamination in laboratory studies [26]. Even highly purified water, such as that obtained from a variety of Milli-Q systems, has been found to contain BPA at concentrations of micrograms per litre [26, 29, 30, 31]. BPA has been detected at concentrations of nanograms per litre in the environment in river waters, sewage run-off, air and soil [25, 26, 32, 33, 34, 35, 36, 37]. Part of the environmental contamination of BPA in our water, air and soil is due to industrial pollution and wastewater run-off from factories that use BPA either in their final product or in the intermediate production stages [12]. Partly as a result of being so ubiquitous in our environment, BPA has been detected in food as well, in the both the canned and fresh varieties [1, 12]. Specifically, BPA has been detected in seafood including fish, prawn, crab, squid and clams [38, 39]. Perhaps most alarming was the finding that waters with no measurable BPA contamination contained fish with BPA present in organs and muscle [38]. This suggests that once taken up, BPA can be stored and concentrated over time in animals. Over the last couple of years, public awareness about BPA has been increasing. Plastic water bottles have begun to disappear off the shelves, starting with Nalgene’s voluntary change to the production of stainless steel water bottles in 2008. Canada has also banned BPA from baby bottles in 2008 in an effort protect infants, as the developing organism is usually more susceptible to environmental toxins than adults. However, the detection of BPA in infant milk formula, as well as human breast milk, demonstrate that this is not enough to protect newborns [23, 40]. These findings highlight a very important point that the general public, although more knowledgeable about BPA than expected, have nonetheless missed. The exclusion of BPA from our immediate usage of food and drink containers does not mean that BPA is absent from our surroundings. Due to its high usage in the plastics and production industries, BPA has become an ubiquitous environmental xenoestro-  3  gen.  1.1.5  Human Exposure to BPA  BPA has been detected in the human fluids blood and urine [26, 23]. While concentrations of BPA measured in humans appear low and average nanograms-micrograms per liter in urine and picograms-nanograms per milliliter in blood, BPA is detectable in the urine of 95-99% of Americans studied [22, 23, 26, 41, 42, 43]. Studies looking at the exposure of BPA in human pregnant women have confirmed the presence of BPA in maternal and fetal plasma, as well as placental tissue at similar concentrations [44, 45]. These studies raise more causes for concern, as they demonstrate the ability of BPA to cross the placental barrier. BPA can also cross the blood-brain barrier, and so has the potential to influence the development of the prenatal brain [46]. After birth, the infant can still be exposed to BPA through the mother, as BPA is secreted in breast milk at concentrations of 0.28-0.87 ng/mL (1.2-3.8nM) [40]. The US Environmental Protection Agency has set a maximum acceptable dose of BPA for humans at 50µg per kilogram bodyweight per day (µg/kg/day) and the European Union at 10µg/kg/day [12]. Human studies suggest a maximum overall exposure to BPA of 1µg/kg/day [12]. Infants were estimated to have a potential BPA exposure of 0.1µg/kg/day from breast milk alone, while formula-fed infants were estimated to have a higher exposure of 1.7µg/kg/day at 3 months of age [40, 47]. The highest exposure to infants were estimated to be at 6-12 months of age, with an average BPA exposure of 13µg/kg/day. While these numbers seem low by comparison to the daily tolerable limit set by governing institutions, it is important to note that the danger of BPA exposure may not lie solely in the concentration. Our continuous exposure to BPA at low concentrations may pose the bigger risk factor, as under these conditions BPA could potentially change the baseline and/or nature of homeostatic mechanisms in our body. In the human population, high concentrations of BPA in urine has been associated with increased risk of cardiovascular disease including angina, coronary heart disease, myocardial infarction and heart attack, as well as diabetes, obesity and recurrent miscarriage [48, 49, 50, 51]. High urinary BPA concentrations are also associated  4  with abnormally high alkaline phosphatase and lactate dehydrogenase liver enzymes, the inflammation marker C-reactive protein and the oxidative stress markers malondialdehyde and 8-hydroxydeoxyguanosine [49, 52, 53]. Because the mechanism of action of BPA has not been completely elucidated, it is important to note that these were correlational studies. However, recent studies have begun to unravel the mechanism behind BPA-induced hyperinsulinemia, which may contribute to the rising incidence of Type II diabetes in the human population [54, 55].  1.1.6  In Vitro Studies  Exposure to high concentrations of BPA (above 400µM) results in neuronal death in the mouse hippocampal cell line HT22 and primary cortical neuron cultures [5, 7, 56]. At these concentrations, neuronal death is induced through apoptosis via activation of Caspase 3 [56]. BPA also increases intracellular calcium, most likely by generating reactive oxygen species, and acts on cell signaling mechanisms to phosphorylate ERK and JNK and induce nuclear translocation of NF-κB [56, 57, 58]. At sublethal concentrations of BPA, Lee et al (2007) reported morphological changes in rat cortical neuron cultures. They described neurite shrinkage, as well as decreased nuclear size, nuclear fragmentation and chromosomal condensation in cells after 24h exposure to BPA. However, at concentrations below 10µM BPA, they reported increased cell viability and neurite extension [5]. Although BPA has some non-estrogen-like effects, experiments have also demonstrated that BPA is similar to estradiol in its effective concentration of action and influences on the brain. In primary cerebellar granule neurons, BPA decreased cell viability to a degree comparable to estradiol as measured by the lactate dehydrogenase assay [7, 59]. In the MCF-7 (breast cancer) and HEK293 (human embryonic kidney) cell lines, BPA induces cell signaling mechanisms and cellular proliferation that are similar to estradiol in effect and concentration [9, 60]. However, BPA’s estrogen-like properties are not inhibited by the presence of the estrogen receptor α antagonists ICI 182,780 or tamoxifen [7, 9, 59, 60]. In the pituitary tumor cell line GH3/B6/F10, which expresses high levels of estrogen receptor α, BPA displayed similar effects as estradiol at picomolar concen-  5  trations by increasing intracellular calcium content [61]. The removal of extracellular calcium extinguished BPA’s ability to increase internal calcium concentrations, suggesting that BPA can also act on voltage dependent calcium channels in the plasma membrane [58, 61]. In other situations, BPA is able to antagonize some of estradiol’s effects in the brain. For example, BPA inhibits estradiol-induced synaptogenesis in the CA1 region of the hippocampus [62]. However, in the CA3 area of the hippocampus, BPA can have effects similar to estradiol on the induction of spine formation [63]. Low, non-neurotoxic concentrations of BPA can have many different effects on different cell types. These effects can follow and mimic those of estradiol, or they can be in direct opposition to estradiol by antagonizing the effects of endogenous or exogenous estrogens [5, 56, 7, 59, 9, 60]. Few, if any, of these effects appear to be inhibited by estrogen receptor antagonists, but some of these effects are also independent of the activation of estrogen receptors [7, 9, 59, 60]. However, because the specifics of the receptor-independent mechanism of action of estradiol remains largely unknown, it is difficult to elucidate these mechanisms from the literature.  1.1.7  In Vivo Studies  Extensive research has been conducted on the effects of BPA on the behavior and neurochemistry of mice and rats. The effects of feeding BPA to rodents have ranged from changes in sexual anatomy, to behavior, to differences found in sexually dimorphic areas of the brain, to no differences being found at all between controls and BPAexposed rodents [64, 65]. Much of these reported and contradicting differences can be traced to the different strains of mice and rats used in the studies, as there are differences in sensitivity to estrogen and BPA between strains of rodents [64]. In addition, within the same rodent strains, the breeding centre of origin can influence the effects of BPA on the organism [64]. It has been suggested that this additional variable is due to differences in diet chow possibly contaminated by phytoestrogens [64]. Nagao et al in 1997 and 2002 found changes in seminal vesicle weight and prostate weights in inbred C57BL/6N and outbred CF-1 mice when dams were exposed to BPA by gavage through gestational days 11-17 [66, 67]. Similar results were  6  also found in outbred Long Evans male rats [68]. In Long Evans rats, gavaging with BPA also resulted in decreased serum 17β-estradiol levels [68]. Exposure of immunodeficient CD-1 pregnant mice during late gestation to BPA through feeding resulted in increases in relative prostate weight, anogenital distance and androgen receptor binding activity, in addition to a decrease in epididymis weight in male offspring [64, 69]. It also increased prostate proliferation, as well as duct number and volume [70]. In outbred Sprague Dawley rats, exposure of the dam to BPA by gavage through gestation resulted in decreased estrogen receptor β staining in the uterine tissue of female offspring [71]. Exposure of lactating dams to BPA also resulted in decreased estrogen receptor α staining in the arcuate nucleus, an area that regulates reproductive and maternal behavior [21, 72]. Della Seta et al (2006) found changes in juvenile investigative behaviors in male Sprague Dawley rats, as well as a decrease in testosterone levels, following BPA exposure through gavage at post natal days 23-30 [73]. Male CD1 mice who were exposed to BPA through dam ingestion during gestation also demonstrated feminized behaviors in free-exploratory open field tests, while females exposed during gestation showed masculinized behaviors in the pre-puberty novelty, free-exploratory open field and elevated plus maze tests [21, 74]. In C57BL/6 mice, exposure of dam to BPA through gavage during gestation resulted in early puberty and increased anxiety in female offspring [75]. In CD-1 dams, gestational, as well as maternal exposure to BPA, decreased nursing time and increased resting time compared to control dams [74]. In female Sprague Dawley rats, gavage with BPA during either gestation or pregnancy also decreased the amount of grooming time dams spent on pups [21, 76]. Interestingly, exposure to BPA in fetal life or gestation changed maternal behavior, but exposure to BPA in fetal life followed by later exposure during gestation did not change maternal grooming of pups [21, 64, 74]. However, other research groups have also found a lack of phenotype associated with BPA exposure [77]. Much of these studies compared the xenoestrogenic effects of BPA to strongly estrogenic positive controls such as the endogenous form of estrogen, 17β-estradiol, or ethinyl estradiol, an oral contraceptive [77]. The lack of a positive estrogenic control does not completely invalidate the literature presented because the 7  strain of mice or rat, the origin of the strain and the behavioral tests used to assess BPA influence on development vary so much from study to study. In addition, while BPA is a weak xenoestrogen, it also possesses other non-estrogenic properties [5, 78]. Studies focussing purely on the estrogenic effects of BPA will miss these other influences, especially when compared to strong estrogens.  1.1.8  Non-Monotonic Dose Response  One of the many complexities of hormone-like signaling properties that BPA possesses is the non-monotonic dose response, also known as hormesis. Hormesis is a phenomenon where a response is elicited at low or intermediate dose levels that disappears at higher dose levels [1, 79]. Traditionally, dose response curves were thought to be linear, with a plateau forming at very high or very low concentrations, where the response of the chemical is maxed out or non-existent, respectively. Because these models were thought to be dogma, most studies on hormonal chemicals have not tested their effects at a wide range of concentrations [1, 79]. Non-monotonic dose response curves are J-, U- or inverted U-shaped and are becoming more commonly associated with hormonal chemicals [1]. The mechanism behind non-monotonic dose responses is also unknown. Despite this, researchers have put forward several theories explaining hormesis. One theory suggests that there is down-regulation of receptors at higher concentrations of hormetic chemicals, while another proposes that hormesis is due to a balancing act between two or more non-synergetic pathways within a cell that act on the same endpoint [1]. In the case of BPA, hormesis may well be the result of the integration of different pathways with different effects at different temperatures on the same cellular or phenotypic output [80]. BPA has genomic and non-genomic effects in the cell, and also appears to bind to a variety of estrogen and androgen receptors with differing affinities [1, 61, 64]. In addition to mimicking estradiol’s effects, BPA also possesses non-estrogenlike properties of its own, such as changing neuron morphology or antagonizing estradiol’s synaptogenic effect in the hippocampus [5, 62]. BPA’s estrogenic effects are not inhibited by estrogen receptor antagonists [7, 9, 59, 60]. Although most  8  studies did not examine whether or not these antagonists also abolish estrogen’s effects on the cell, it highlights the fact that BPA has estrogen-receptor dependent and independent effects [78]. Some of BPA’s non-monotonic dose responses in cells include: activation of extracellular signal-regulated kinase in cerebellar neurons, proliferation of LNCap prostate and MCF-7 breast cancer cells, changes in neurite length in PC12 and cortical neurons, increase in intracellular calcium from the extracellular milieu in GH3/B6 pituitary cells, inhibition of adiponectin secretion from adipose tissue and release of insulin from pancreatic islet cells [1, 5, 59, 81, 82, 83, 84, 85]. In light of these studies, suggestions that BPA as an environmental contaminant is safe merely because environmentally irrelevant high dosages are required to cause cell death or dysfunction would be premature. The hallmark of hormesis is that very low concentrations can produce effects that disappear at higher concentrations. Relying purely on traditional toxicological methods of short exposure times at high concentrations would completely miss the potentially alarming consequences of lower concentrations.  1.1.9  Controversies  Many of the studies discussed in the previous three sections were carried out in academic research labs. Because BPA is so widely used in the plastics industry and so widely incorporated in the production of everyday materials, there is great criticism and great resistence to the acknowledgement of BPA as a potentially harmful xenoestrogen from the plastics industry. This resistence partly stems from the inability of industry to replicate academic research on BPA, as well as industry experiments that show BPA to have very little to no estrogenic or other negative effects in mice and rats [86, 87]. In contrast, very few academic studies have found BPA to be harmless according to the variables measured [77]. Academic researchers, too, have been unable to replicate industry findings, and have also expressed great criticisms against the controls and measures used by plastics industry. Recently, BPA researchers have published a collaborative commentary protesting against the government’s insistance on dismissing reports of BPA’s effects on  9  health and behavior [88]. These researchers have now banded together in an effort to standardize experimental techniques and dosages of BPA and comply with industry criticisms to determine the actual effects of BPA on mammals [89]. These are important steps because in addition to deciding the potential harmfulness of environmental exposures to BPA, the results, methodologies and understanding of industry standards have potential to change the way future toxicological studies will be conducted.  10  1.2  Sterol Regulatory Element Binding Protein 1  Sterol regulatory element binding protein 1 (SREBP 1) is a transcription factor normally sequestered as a 125kDa transmembrane protein anchored to a SREBP cleavage-activating protein (SCAP) in the endoplasmic reticulum [90]. Under conditions of low sterol abundance in the cell, SREBP 1 is cleaved by the membrane-bound enzyme Site-1 protease (S1P), which recognizes the SREBP 1/SCAP complex (Figure 1.4 [90, 91, 92]. Next, the transmembrane enzyme Site-2 protease (S2P) cleaves one of the SREBP 1 portions at the cytosolic side to form a 68kDa free protein, which then translocates to the nucleus to act as a transcription factor [90, 93]. There are three isoforms of SREBP 1: SREBP 1a, SREBP 1c and SREBP 2 [90]. All 3 isoforms have two transmembrane regions and are situated in the endoplasmic reticulum such that both the carboxyl and amino terminals are cytosolic, with a lumenal loop. The carboxyl terminal is the regulatory region that interacts with SCAP and S1P [90]. Upon cleavage, the amino terminal functions as a transcription factor [90].  1.2.1  Functions of SREBP 1  In the nucleus, SREBP 1 regulates the transcription of enzymes involved in cholesterol, fatty acid, triglyceride and phospholipid biosynthesis, secretory proteins including the hormone leptin, proteins that participate in lipid catabolism and intracellular transport, G-proteins and voltage gated ion channels [94, 95, 96, 97, 98, 99]. The fluidity of the plasma membrane and the presence of voltage gated ion channels are of great importance in signaling neurons, as these factors directly influence the ability of the cell to signal and release neurotransmitters. As a result of its regulation of lipid biosynthesis, research on SREBP 1 has focussed primarily on the roles and regulation of this transcription factor in the liver and hepatocytes. It is only recently, with the discoveries that SREBP 1 is activated in many models of cell death that investigations into the role of SREBP 1 in the brain have begun. In non-neuronal cells, SREBP 1 is activated in glucolipotoxic cell death in pancreatic β cells, as well as anaerobic, hypotonic and hypoxic stress-induced cell 11  death in yeast, Chinese Hamster Ovary cell lines and C. elegans [54, 96, 100, 101, 102, 103, 104, 105]. Last year, Taghibiglou et al (2009) reported that the activation of SREBP 1, but not SREBP 2, in NMDA-induced excitotoxic stress led to neuronal cell death. Inhibition of SREBP 1 activation rescued these neurons from cell death, as did calcium ion chelators [96]. BPA also increases intracellular calcium concentrations [56, 58]. This suggests that BPA may activate SREBP 1 through calcium influx. If so, then findings that BPA exposure is correlated positively with increased plasma insulin, risk of diabetes and obesity in Swiss albino OF1 mice, may have a cause and effect relationship [49, 55]. This hypothesis in neuron cultures is examined in chapter 4.  12  1.2.2  Model System  The model system utilized in our studies is that of embryonic Wistar rat cortical neurons grown in dissociated culture. Briefly, cortical neurons were harvested from the cortex of embryonic day 18 Wistar rats, without distinction between the different cortices. These neurons were trypsinized and plated in dissociated form on poly-Dlysine coated plates in Neuralbasal media. Wistar Rat Cortical Neurons Cortical neurons grown in a dissociated culture system acquire morphological, electrophysiological and pharmacological features similar to that of the in vivo cortex, and as such, comprises a resource-efficient way of determining the degree of neurotoxicity of BPA in the in vivo situation [106]. This method of determining BPA neurotoxicity also allowed us to specifically determine the concentrations of BPA that the neurons are exposed to. In in vivo studies of BPA neurotoxicity, the exposure route of BPA is through gavage, and the penetration of BPA into the organism is determined through measurement of concentrations in blood as well as radioactive-labelled BPA [43, 107]. However, the exact concentrations of BPA that the brain is exposed to is unknown. In addition, assuming the pentrance of BPA into cerebrospinal fluid, neurons in the deeper layers of the brain as compared to the superficial layers are likely to be exposed to different concentrations of BPA over time as it is metabolized or broken down, which adds a layer of complexity to in vivo studies. The growth of cortical neurons in a monolayer, as in our dissociated system, ensures equal exposure of BPA to each neuron. The harvest of cortical neurons gives a bigger yield per embryonic mouse as compared to the harvest of other neuronal types. In addition to reducing the number of animals to be sacrificed to answer our experimental questions, cortical neurons also make up the bulk of the neurons in the brain. Therefore, any changes observed in the health status or function of cortical neurons are likely to have an effect at the behavioral level in the organism. The effects of BPA on rat cortical neurons were measured at DIVs 9 and 15 because we were interested in the comparison of the effects of BPA on synaptically  13  immature (DIV9) versus synaptically mature neurons (DIV15) [108, 109, 110]. At the receptor level, there are differences in NMDA receptor subunit expression between DIV9 and DIV15. While NR1 and NR2B receptor subunits are expressed at increasing levels starting after DIV1, NR2A expression does not start to increase until around DIV9 [110]. At DIV15, while NR1 and NR2A receptor expression levels are still increasing, NR2B receptor expression is starting to plateau off [110]. As a result of these differences in NMDA receptor subunit expression, we decided to look at the effects of BPA on neurons at different developmental stages as delineated by subunit expression to elucidate the possible influence of synaptic receptor type and maturity on BPA impact on neuronal health.  14  1.3  Aims of Thesis  The aims of this thesis are to: 1. Determine the effects of BPA on the viability of rat cortical neuron cultures as a model of the effects of BPA on the human pre- or perinatal brain. In chapter 2, we investigated the effects of different concentrations of BPA ranging from 1mM to 10fM on the viability of cortical neuron cultures under acute and chronic exposure conditions. Additionally, we examined the viability of cultures treated with 10pM BPA at 1, 3, 5 and 9 hours (h) of exposure. We hypothesized that BPA will decrease neuronal viability differently under acute and chronic exposure conditions. 2. Determine the effects of BPA on the response of rat cortical neuron cultures to oxidative or excitotoxic stress as a model of the effects of BPA on the human pre- and perinatal brain. In chapter 3, we examined the effects of acute or chronic BPA exposure on the ability of rat cortical neuron cultures to survive an oxidative or excitotoxic stress challenge. We also studied the viability of cultures pre-treated with 10pM BPA at 1, 3, 5 and 9h of exposure prior to oxidative or excitotoxic stress onslaught. We hypothesized that neuronal survival following an oxidative or excitotoxic stress challenge will be compromised by BPA exposure under both acute and chronic conditions. 3. Determine the effect of BPA on cell regulation mechanisms inside neurons by measuring the activation of SREBP 1 in response to BPA exposure. In chapter 4, we examined the effects of different concentrations of BPA on the activation of SREBP 1 after 5 hours of exposure. We hypothesized that BPA will change the activation of SREBP 1 in a concentration-dependent manner in cortical neuron cultures.  15  Figure 1.1: Structure of Bisphenol A. It consists of two phenol rings linked together by a methyl bridge with two methyl groups. BPA is able to bind to estrogen receptors α and β, membrane-bound and transmembrane G protein-couled estrogen receptor, orphan nuclear receptor and aryl hydrocarbon receptor. Figure obtained from Rubin and Soto (2009).  16  Figure 1.2: Structure of 17β-estradiol. Note the phenol group that is also present in BPA and genistein, a phytoestrogen. Figure obtained from Rubin and Soto (2009).  17  Figure 1.3: Structure of genistein, a phytoestrogen. Note the phenol group that is also present in 17β-estradiol and BPA. Figure obtained from Badeau (2008).  18  Figure 1.4: SREBP 1 activation as a transcription factor through cleavage by S1P and S2P. The regulatory domain of SREBP 1 is at the carboxyl terminal, while the amino terminal contains the recognition sequence for DNA binding. Top: SREBP 1 is found in a complex with SCAP. Sterols normally inhibit S1P from cleaving SREBP 1, but under sterol-poor conditions, S1P recognizes the SREBP 1-SCAP complex and cleaves SREBP 1 at the lumenal loop. Middle: After cleavage by S1P, S2P is able to recognize and cleave SREBP 1 at the cytosolic side. Bottom: The free amino terminalend of SREBP 1 translocates to the nucleus, where it binds to DNA and activates the transcription of genes. Figure obtained from Brown and Goldstein (1999).  19  Chapter 2 Effects of Acute and Chronic BPA Exposure on Neuronal Health 2.1  Introduction  BPA is known to be neurotoxic at concentrations around 100µM (22.839µg/mL) [5, 7, 56]. In line with the main views in toxicology, most studies have looked at short incubation times with barely sublethal concentrations of BPA [56]. However, BPA has a non-monotonic response dose curve [23]. In hormonal chemicals with nonmonotonic response dose curves, it is not always the concentration of the chemical that determines its magnitude of response, but the time of exposure prior to measurement as well [79]. BPA is also an environmental xenoestrogen that is most likely present at very low concentrations in our body for very long periods of time. Therefore, it is likely that markedly different exposure times to BPA will have different effects on neuronal viability and health.  2.1.1  MTT Assay  The MTT assay consists of incubating a yellow, water soluble 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide (MTT) compound in cell culture medium for four hours to overnight to allow for complete reduction of MTT by mitochondrial dehydrogenase [111, 112, 113]. This chemical reaction changes yellow MTT into a purple, water-insoluble formazon [111, 112, 113]. Purple formazon can be dissolved in 20  dimethyl sulfoxide (DMSO, Sigma-Aldrich, Saint Louis, MO) [112, 113]. The amount of formazon reduced can be determined spectrophotometically at 570nm [113]. Because reduction of MTT only occurs in living cells, the quantity of formazon produced is proportional to the number of viable cells in culture [112, 113]. Thus, comparing values of absorbance between treatments can give a qualitative measure of culture viability across treatments within the same experiment [112, 113].  2.1.2  Aims of Chapter  In this chapter, we examine the effects of different concentrations of BPA on primary rat cortical neuron cultures under acute (5h) and chronic (DIV3-9 or DIV3-15) conditions. Much of the work done on BPA in the literature focussed on short periods of exposure to very high concentrations of BPA, but this is not representative of human exposure to BPA. We hypothesize that at very high (1mM) and very low (100fM) concentrations of BPA, acute and chronic exposure will give a similar phenotype on our neuronal cultures. However, at immediately sublethal concentrations of BPA, we hypothesize that there may be a difference in neuronal viability between the acute and the chronic exposures.  21  2.2  Materials and Methods  2.2.1  Primary Rat Neuron Culture and Maintenance  Primary rat cortical neurons were harvested from gestational day 18 (e18) embryos. The pregnant Wistar rat, obtained from Charles River (Charles River, Senneville, Quebec) and later CDM (Centre for Disease Modeling, Vancouver, British Columbia), was anaesthesized with an intraperitoneal injection of urethane and the embryos were transferred onto a plate of pre-chilled dissection buffer (0.015M HEPES, 0.015M sucrose and 0.111M glucose in Hank’s Balanced Salt Solution (HBSS), pH adjusted to 7.37-7.43 and osmolarity 310-320mOsm). HBSS, HEPES, sucrose and glucose were obtained from Sigma-Aldrich, Saint Louis, MO. The cortices were separated and trypsinized with a 0.25% trypsin-EDTA solution (Gibco-BRL, Grand Island, NY) for 30 minutes (min) at 37◦ Celcius. After removal of trypsin by three washes with prewarmed 10% fetal bovine serum (FBS, Gibco-BRL, Grand Island, NY) in Dulbecco’s Modified Eagle Medium (DMEM, Gibco-BRL, Grand Island, NY), the cells were mechanically trypsinzed by three passages through a 1mL pipette tip.The suspension was then spun down and the supernatant discarded. The cell pellet was resuspended in Neurobasal medium supplemented with 1x B27, 0.5mM GlutaMAX, 50units/mL penicillin and 500µg/mL streptomycin (all obtained from Gibco-BRL, Grand Island, NY). At least 18h prior to neuron harvest, 24-well tissue culture plates (Corning Inc) were coated with a poly-D-lysine solution (5mg in 500mL filtered 0.15M sodium tetraborate decahydrate solution at pH8.4, both obtained from Sigma-Aldrich, Saint Louis, MO). At least 12h post-coating, the poly-D-lysine solution was removed and tissue culture plates were washed three times with autoclaved distilled water. Cortical cells were plated at a concentration of 1.25x105 per well in a 24-well tissue culture plate. Cells were maintained at 37◦ Celcius, 5% CO2 humidity in the incubator (NuAir, Plymoouth, MN). Half of the media was replaced once to twice weekly.  22  2.2.2  BPA Treatment  BPA (Sigma-Aldrich, Saint Louis, MO) does not dissolve well in distilled water and so was dissolved in 100% ethanol (EtOH, UBC Chemistry Stores) to obtain a stock concentration of 1M. Prior to neuron treatment, the stock solution of BPA was diluted in culture media and then added to each well at a 1:100 dilution to the desired concentrations. This solution was stored at -20◦ Celcius. 100% EtOH was diluted in culture media at a 1:10 then added to neuronal culture at a 1:100 dilution as a vehicle control. This resulted in a final EtOH concentration of 0.1%.  2.2.3  MTT Assay  A 12mM MTT solution was prepared by dissolving 3-(4,5-Dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (Sigma-Aldrich, Saint Louis, MO) in sterile phosphate buffered saline solution (PBS) at 5mg:1mL. This solution was kept at 4◦ Celcius and protected from light. At the conclusion of each BPA treatment, cultures were incubated with 50µL of MTT solution for 24h. 24h post-MTT addition, culture media was aspirated and 220µL of a 10% glycine (Bio-Rad, Hercules, CA) buffer in DMSO solution was added to dissolve the precipitated formazon. The absorbance of each sample at 570nm was recorded in a spectrophotometer (Bio Tek Instruments, Inc., Burlington, VT) and subtracted from the absorbance of a blank composed of a 10% glycine buffer in DMSO solution [113].  2.2.4  Statistical Analysis  Statistical analyses were performed using Instat+ software (Statistical Services Centre, The University of Reading, United Kingdom). Treatment effects were analyzed with one-way analysis of variance (ANOVA), followed by post hoc Tukey Honest Significant Difference (HSD) Test. Significance was set at at p <0.05.  23  2.3  Study Design  Acute BPA Exposure with Concentration Variation Primary rat cortical neurons were plated in 24-well tissue culture plates. At DIV15, BPA was added to each well to final concentrations of 1mM (0.228mg/mL), 500µM (0.114mg/mL), 10µM (2.283µg/mL), 10nM (2.283ng/mL), 10pM (2.283pg/mL) and 10fM (2.283fg/mL). 5h post-BPA addition, MTT solution was added to each well. Cells were incubated with MTT solution for at least 4h-overnight to measure overall cell viability. Acute 10pM BPA Exposure at with Time Variation Primary rat cortical neurons were plated in 24-well tissue culture plates. At two timepoints, DIV9 and DIV15, BPA was added to each well at timepoints 0, 4, 6 and 8h to a final concentration of 10pM. At 9h, MTT solution was added to each well to obtain total BPA incubation times of 9, 5, 3 and 1h respectively. Post-MTT addition, cells were incubated for an addition 4h-overnight to measure overall cell viability. Chronic BPA Exposure Primary rat cortical neurons were plated in 24-well tissue culture plates. At DIV3, 100% of the culture media was replaced with fresh media supplemented various concentrations of BPA. Half of the culture media was replaced with fresh media supplemented with BPA twice a week. At DIV9, MTT solution was added to wells containing neurons exposed to the following concentrations of BPA: 10µM (2.283µg/mL), 100nM (22.829ng/mL), 1nM (0.228ng/mL), 10pM (2.283pg/mL), 100fM (22.829fg/mL) and 1fM (0.228fg/mL). At DIV14-16, MTT solution was added to wells containing neurons exposed to the following concentrations of BPA: 1mM (0.228mg/mL), 500µM (0.114mg/mL), 100µM (22.829µg/mL), 10µM (2.283µg/mL), 100nM (22.829ng/mL), 1nM (0.228ng/mL), 10pM (2.283pg/mL), 100fM (22.829fg/mL) and 1fM (0.228fg/mL). The cells were incubated with MTT solution for 4h-overnight to measure overall cell viability.  24  2.4 2.4.1  Results Effect of Acute BPA Exposure on Cortical Neuron Health  Acute BPA Exposure with Concentration Variation The exposure of DIV15 primary rat cortical neurons to 1mM BPA for 5h resulted in a significant 25% decrease in cell viability compared to the media control (NB in figure 2.1) as detected by a MTT assay. Treatment of DIV15 neurons with 500µM (0.114mg/mL), 10µM (2.283µg/mL), 10nM (2.283ng/mL), 10pM (2.283pg/mL) or 10fM (2.283fg/mL) BPA did not significantly decrease cell viability as compared to the media control after 5h as detected by a MTT assay (Figure 2.1). Acute 10pM BPA Exposure at with Time Variation The exposure of DIV9 primary rat cortical neurons to 10pM BPA for 1, 3, 5 or 9h did not significantly change cell viability as detected by a MTT assay (Figure 2.2). There was no apparent decrease nor increase in cell viability as compared to the negative (0h) and the vehicle controls. The exposure of rat cortical neurons grown to DIV14-16 to 10pM (2.283pg/mL) BPA for 1, 3, 5 or 9h also did not significantly change cell viability as compared to the negative (0h) and the vehicle controls (Figure 2.3).  2.4.2  Effect of Chronic BPA Exposure on Cortical Neuron Health  The viability of neurons grown in culture were measured by the MTT assay at DIV9 and DIV14-16. Chronic exposure of rat cortical neurons to various concentrations of BPA began at DIV3 with a complete media change until assessment by MTT. Only 1mM (0.228mg/mL) and 500µM (0.114mg/mL) concentrations of BPA caused 85% and 90%, respectively, neuronal death in the cultures, as measured by a significant decrease in the formation of formazon (Figures 2.4 and 2.5). Exposure of cortical neurons to concentrations of BPA ranging from 100µM (22.829µg/mL) to 1fM (0.228fg/mL) from DIV3-9 or DIV14-16 did not cause a significant change in  25  neuronal death or metabolism as measured by the MTT assay.  26  2.5  Discussion  In this study, the effects of concentrations of BPA ranging from 1mM (0.228mg/mL) to 1fM (0.228fg/mL) on the viability of primary rat cortical neurons grown in culture were examined in both the acute and the chronic situation. When DIV15 neurons were treated with BPA under the acute condition, we found that exposure to 1mM (0.228mg/mL) BPA resulted in measurable cell death after 5h. Exposure to 1mM (0.228mg/mL) and 500µM (0.114mg/mL) BPA under the chronic condition, from DIV3 to DIV15, resulted in more extensive neuronal cell death as based on MTT assays. This corresponds well to a study by Lee et al (2008) where exposure of HT22 cells (a mouse hippocampal cell line) to 500µM (0.114mg/mL) BPA over 24h resulted in a decrease in cell viability. While exposure to 500µM (0.114mg/mL) BPA did not cause cell death in the acute condition in this study, our exposure time was only 5h long, while Lee et al (2008) had a 24h long exposure time [56]. At exposure times greater than 5h, 1mM (0.228mg/mL) and 500µM (0.114mg/mL) BPA decreased cell viability by more than 85% (Figure 2.5). Concentrations of BPA below 500µM (0.114mg/mL) were not found to have any neurotoxic effect, nor did they significantly increase cell viability (Figure 2.1), which supports and contradicts the findings of Lee et al (2008) respectively. However, the studies conducted by Lee et al (2008) were on a neuronal cell line, while our studies were conducted on primary neurons. The response of cell lines to stimulants can differ from that of primary cultures, due to changes in signaling pathways. Our results also support those of Kim et al (2008), whose work in neural progenitor cells described cell death at 500µM (0.228mg/mL) BPA [57]. Furthermore, Kim et al (2008) showed that micromolar concentrations of BPA markedly increased the level of reactive oxygen species in neural progenitor cells after 12h. Intraperitoneal injection of BPA into mice has been reported to result in increased hydrogen peroxide production in the kidneys, liver, testis and brain, further supporting this conjecture [114]. The production of reactive oxygen species could be a method by which 500µM (0.114mg/mL) BPA caused neuronal cell death that was only measurable under the chronic condition in our study, as the 5h study design in the acute experiment may 27  not have been sufficient to allowed reactive oxygen species to cause cell death. Under the acute condition, although 500µM (0.228mg/mL) BPA did not cause a decrease in cell viability, it did slightly increase cell viability as measured by a MTT assay (Figure 2.1). This slight increase may be due to increased metabolism or increased mitochondrial function in the cell. Under the chronic condition, incubation of neurons with 10µM (2.283µg/mL) BPA from DIV3-9 resulted in a slight increase in cell viability (Figure 2.4). While this increase is not statistically significant different from the control, it may have been due to unequal media evaporation across the culture plate, as the placements of each treatment condition were not randomized across replications in this experiment. We suspect that this is the correct explanation, as the increase is not very marked (only about 15% more compared to control), and the same trend was not seen under the acute condition (Figure 2.1), nor under the chronic condition in which BPA incubation lasted from DIV3-15 (Figure 2.5). An alternative explanation is the suggestion of Lee et al (2008) where they hypothesized that doses of BPA below 50µM (11.415ug/mL) could result in neurite extension, estrogen receptor activation and neuroprotection [56]. Increased neurite extension could increase the metabolic output of a cell, resulting in a measure of increased cell viability with the MTT assay. However, in this study, we did not look at morphological changes in our cultures in response to BPA treatment. While 10pM (2.283pg/mL) BPA did not cause neuronal death under the acute and chronic conditions (Figures 2.1, 2.4 and 2.5), we looked specifically at the effect of 10pM (2.283pg/mL) BPA on DIV9 and DIV15 neuron viability at 1, 3, 5 and 9h (Figures 2.2 and 2.3). This was due to the eperiments we were, at the time, concurrently conducting with SREBP 1 in which we saw maximal effect at picomolar concentrations of BPA (see chapter 4). We had hypothesized that increased activation of SREBP 1 may cause neuronal cell death or susceptibility in a time-dependent manner that had its maximal effect shortly after 5h but neuronal viability did not change in response to 1, 3, 5 or 9h exposure time to 10pM (2.283pg/mL) BPA [96].  28  Figure 2.1: Effect of 5h acute BPA treatment on the viability of DIV15 neurons. e18 primary Wistar rat cortical neurons were plated in 24-well tissue culture plates at 1.25x105 cells per well. At DIV15, BPA was added to each well to final concentrations of 1mM (0.228mg/mL), 500µM (0.114mg/mL), 10µM (2.283µg/mL), 10nM (2.283ng/mL), 10pM (2.283pg/mL) and 10fM (2.283fg/mL). 5h post-BPA addition, 50µL MTT solution was added to each well and the cells were incubated for a further 24h. Precipitated formazon was dissolved in a solution of 10% glycine in DMSO and the absorbance of each sample at 570nm was recorded in a spectrophotometer. Cell viability is expressed as relative absorbance compared to control (NB). Treatment effects were analyzed with one-way ANOVA, followed by the post-hoc Tukey HSD test. The results are presented as means ± SEMs of 3 independent experiments; N=(from left to right) 7, 5, 5, 4, 5, 4, 4, 4; p <0.05 (* indicates statistical significance from control).  29  Figure 2.2: Effect of acute 10pM BPA treatment on the viability of DIV9 neurons. e18 primary Wistar rat cortical neurons were plated in 24-well tissue culture plates at 1.25x105 cells per well. At DIV9, BPA was added to each well at timepoints 0, 4, 6 and 8h to result in incubation times of 9, 5, 3 and 1h respectively. The final concentration of BPA in each well was 10pM (2.283pg/mL). At the 9h timepoint, 50µL MTT solution was added to each well and the cells were incubated for a further 24h. Precipitated formazon was dissolved in a solution of 10% glycine in DMSO and the absorbance of each sample at 570nm was recorded in a spectrophotometer. Cell viability is expressed as relative absorbance compared to control (NB). Treatment effects were analyzed with one-way ANOVA. The results are presented as means ± SEMs of 3 independent experiments; N=3 for all treatments shown; p <0.05. No statistical significance from control (NB) was found.  30  Figure 2.3: Effect of acute 10pM BPA treatment on the viability of DIV15 neurons. e18 primary Wistar rat cortical neurons were plated in 24-well tissue culture plates at 1.25x105 cells per well. At DIV15, BPA was added to each well at timepoints 0, 4, 6 and 8h to result in incubation times of 9, 5, 3 and 1h respectively. The final concentration of BPA in each well was 10pM (2.283pg/mL). At the 9h timepoint, 50µL MTT solution was added to each well and the cells were incubated for a further 24h. Precipitated formazon was dissolved in a solution of 10% glycine in DMSO and the absorbance of each sample at 570nm was recorded in a spectrophotometer. Cell viability is expressed as relative absorbance compared to control (NB). Treatment effects were analyzed with one-way ANOVA. The results are presented as means ± SEMs of 4 independent experiments; N=4 for all treatments shown; p <0.05. No statistical significance from control (NB) was found.  31  Figure 2.4: Effect of chronic BPA exposure on the viability of DIV9 neurons. e18 primary Wistar rat cortical neurons were plated in 24-well tissue culture plates at 1.25x105 cells per well. At DIV3, 100% of the culture media was replaced with fresh media supplemented with concentrations of BPA ranging from 10µM (2.283µg/mL)1fM (0.228fg/mL). At DIV9, 50µL MTT solution was added to each well and the cells were incubated for a further 24h. Precipitated formazon was dissolved in a solution of 10% glycine in DMSO and the absorbance of each sample at 570nm was recorded in a spectrophotometer. Cell viability is expressed as relative absorbance compared to control (media). Treatment effects were analyzed with one-way ANOVA. The results are presented as means ± SEMs of 3 independent experiments; N=(from left to right) 4, 6, 5, 6, 6, 6; p <0.05. No statistical significance from the media control was found.  32  Figure 2.5: Effect of chronic BPA exposure on the viability of DIV15 neurons. e18 primary Wistar rat cortical neurons were plated in 24-well tissue culture plates at 1.25x105 cells per well. At DIV3, 100% of the culture media was replaced with fresh media supplemented with concentrations of BPA ranging from 1mM (0.228mg/mL)1fM (0.228fg/mL). Half of the culture media was replaced with fresh media supplemented with BPA twice a week. At DIV15, 50µL MTT solution was added to each well and the cells were incubated for a further 24h. Precipitated formazon was dissolved in a solution of 10% glycine in DMSO and the absorbance of each sample at 570nm was recorded in a spectrophotometer. Cell viability is expressed as relative absorbance compared to control (media). Treatment effects were analyzed with one-way ANOVA, followed by the post-hoc Tukey HSD test. The results are presented as means ± SEMs of 6 independent experiments; N=(from left to right)12, 3, 3, 3, 14, 14, 14, 14, 14, 8; p <0.05 (* indicates statistical significance from control).  33  Chapter 3 Effects of BPA on Neuron Response to Excitotoxic and Oxidative Stress Challenge 3.1  Introduction  Low concentrations of BPA are not neurotoxic, but they change signaling patterns and phosphorylation status of proteins within a cell [56, 57]. In some neuron cell types, such as cerebellar granule cells, it is known that endogenous estradiol is neurotoxic at around 0.1nM [7]. In these cells, BPA mimics the actions of estradiol at similar concentrations [7, 59]. However, it is unclear what the effects of other low concentrations of BPA in primary rat cortical neuron cultures might be. The experiments conducted in chapter 2 have shown that even under chronic exposure to BPA, low concentrations are not neurotoxic and do not significantly change neuronal viaibility as measured by a MTT assay. However, cell death is a very blunt endpoint measure of the effects of a toxin or xenoestrogen. In the in vivo and the in vitro situation, much damage can occur in a cell before the endpoint of death is reached. This damage can occur in the form of morphological changes, activation of apoptotic mechanisms, loss of function, or more simply, an inability to respond to neurotoxic stress [5, 7, 56, 57]. Therefore, it is likely that while markedly different exposure times to BPA  34  may not drastically change neuronal viability, there may be more subtle and insidious effects on neuronal health depending on the concentration and length of exposure. One measure of a negative impact on neuronal health without causing cell death is increased susceptibility to neurotoxic stress, which will result in deceased cell viability.  3.1.1  Neurotoxic Stress Challenge  Two types of neurotoxic stress challenges investigated were excitotoxic and oxidative cell stress. These two specific causes of neurotoxicity have been shown to be involved, or are suspected to play a role in, neuronal degenerative disorders [115]. In addition, these neurotoxic stresses constitute situations that neurons are likely to experience in the in vivo situation, giving in vitro findings real-world implications. Excitotoxic Stress Challenge In both the in vivo and in vitro situations, high concentrations of excitatory neurotransmitters such as glutamate and NMDA in the central nervous system milieu can cause neuronal death and physical degeneration in exposed areas of the brain [116]. The major mechanism behind this type of neuronal cell death is the massive calcium influx from from hyperstimulation of the NMDA receptors by glutamate and NMDA [116]. The uncontrolled influx of calcium, a second messenger molecule, can cause activation and inactivation of various other enzymes in many cell signalling pathways [115]. While excitotoxicity is an apparent problem in patients with epilepsy, high concentrations of glutamate and NMDA have been measured in hypoglycemia and ischemia and may be a cause of neuronal death in these situations [116]. In addition, excitotoxicity is suspected to have a role in the pathogenesis of neurodegenerative disorders such as Huntington’s Disease, Alzheimer’s Disease and amyotrophic lateral sclerosis [115, 116]. As a result, we decided to investigate the effects of BPA on neuronal response to excitotoxic cell stress.  35  Oxidative Stress Challenge One cause of oxidative stress are reactive oxygen species [117]. Reactive oxygen species are formed as a byproduct of or released as a cytotoxic danger to neurons in response to mitochondrial respiration, cell lysis or injury, impairment of detoxification by antioxidants and excitotoxicity [117, 118]. In addition to changing major cell signaling pathways such as the MAPK cascade, oxidative cell stress can also cause apoptotic cell death in neurons [115, 117, 119]. In addition causing neuronal death, oxidative stress may have a role in the pathogenesis of neurodegenerative disorders such as Alzheimer’s Disease, Parkinson’s Disease and amyotrophic lateral sclerosis. In vitro studies of oxidative stress commonly expose hydrogen peroxide, a reactive oxygen species, to the cell culture system being investigated [117].  3.1.2  Aims of Chapter  In this chapter, we examine the effects of different concentrations of and different exposure times to BPA on the ability of primary rat cortical neuron cultures to survive an oxidative or excitotoxic stress challenge. Taking into consideration the results of the experiments presented in chapter 2 and 4, we hypothesize that the ability of our neuron cultures to survive an oxidative or excitotoxic stress challenge will be compromised under a specific concentration and exposure time to BPA.  36  3.2 3.2.1  Materials and Methods Primary Rat Neuron Culture and Maintenance  See section 2.2.2  3.2.2  BPA Treatment  See section 2.2.3  3.2.3  Oxidative and Excitotoxic Stress Challenge  Hydrogen peroxide (H2 O2 , 30% w/v) and N-methyl-D-aspartic acid (NMDA) were obtained from Sigma-Aldrich, Saint Louis, MO. Glycine was purchased from BioRad, Hercules, CA. Stock solutions of 50mM NMDA and 20mM glycine were made in double distilled water and stored at 4◦ Celcius. Prior to each oxidative stress challenge, hydrogen peroxide (Sigma-Aldrich, Saint Louis, MO) was diluted in culture media to 40µM. All chemicals used in the oxidative (hydrogen peroxide) and excitotoxic (NMDA, glycine) stress challenge were diluted 1:10 immediately prior to neuron treatment. Oxidative Stress Challenge The diluted hydrogen peroxide solution was added to each well at 1:100 dilution. The neurons were incubated with hydrogen peroxide at 37◦ Celcius, 5% CO2 for 12h to simulate oxidative stress prior to the addition of MTT solution to measure cell viability/survival. Excitotoxic Stress Challenge Prior to the excitotoxic challenge, 300µL media was removed from each well and stored in the incubator at 37◦ Celcius. The previously diluted NMDA was then added to each well at 1:100 while glycine was added at 1:200, resulting in final concentrations of 50µM NMDA and 10µM glycine per well. The culture plate was returned to the incubator at 37◦ Celcius, 5% CO2 for 1h, after which culture media was aspirated  37  and replaced with the previously removed media (300µL). The culture plate was then returned again to the incubator for 24h to allow for neuron recovery.  3.2.4  MTT Assay  See section 2.2.4  3.2.5  Statistical Analysis  Statistical analyses were performed using Instat+ software (Statistical Services Centre, The University of Reading, United Kingdom). Treatment effects were analyzed with one-way analysis of variance (ANOVA), followed by the post hoc Tukey Honest Significant Difference (HSD) Test. Significance was set at at p <0.05.  38  3.3  Study Design  Effect of Acute BPA Exposure on Neuron Response to Excitotoxic Stress Challenge Primary rat cortical neurons were plated in 24-well tissue culture plates. At DIVs 9 and 15-16, diluted BPA was added to each well to a final concentration of 10pM (2.283pg/mL). This was done at timepoints of 0, 4, 6 and 8h to yield total incubation times of 9, 5, 3 and 1 hours respectively at 9h. At 9h, NMDA and glycine were added into the wells to simulate excitotoxic stress. At 10h, 100% of the media was replaced, and the cells were allowed to recover. After 24h of recovery time, MTT solution was added and the cells were incubated for an addition 4h-overnight to measure cell viability. Effect of Acute BPA Exposure on Neuron Response to Oxidative Stress Challenge Primary rat cortical neurons were plated in 24-well tissue culture plates. At DIVs 9 and 15-16, diluted BPA was added to each well to a final concentration of 10pM (2.283pg/mL) at timepoints of 0, 4, 6 and 9h to yield total incubation times of 9, 5, 3 and 1 hours respectively at 9h. At 9h, hydrogen peroxide was added into the wells to simulate oxidative stress. The cells were then returned to the incubator for 12h, after which MTT was added. The cells were incubated for an addition 4h-overnight to measure cell viability. Effect of Chronic BPA Exposure on Neuron Response to Excitotoxic Stress Challenge Primary rat cortical neurons were plated in 24-well tissue culture plates. At DIV3, 100% of the culture media was replaced with fresh media supplemented with the following concentrations of BPA: 10µM (2.283µg/mL), 100nM (22.829ng/mL), 1nM (0.228ng/mL), 10pM (2.283pg/mL), 100fM (22.829fg/mL) and 1fM (0.228fg/mL). Once-twice weekly thereafter, half of the culture media was replaced with fresh media supplemented with BPA. At DIV14-16, NMDA and glycine were added into the wells  39  to simulate excitotoxic stress. 1h post-addition, 100% of the media was replaced with either fresh Neurobasal or preconditioned media. The plates were returned to the incubator for 24h before the addition of MTT solution. Effect of Chronic BPA Exposure on Neuron Response to Oxidative Stress Challenge Primary rat cortical neurons were plated in 24-well tissue culture plates. At DIV3, 100% of the culture media was replaced with fresh media supplemented with the following concentrations of BPA: 10µM (2.283µg/mL), 100nM (22.829ng/mL), 1nM (0.228ng/mL), 10pM (2.283pg/mL), 100fM (22.829fg/mL) and 1fM (0.228fg/mL). Once-twice weekly thereafter, half of the culture media was replaced with fresh media supplemented with BPA. At DIVs 9 and 14-16, hydrogen peroxide was added into the wells to simulate oxidative stress and the cells were returned to the incubator. 12h post-addition, MTT solution was added and the cells were incubated for an additional 4h-overnight before the formazon was dissolved and the absorbance read.  40  3.4  Results  3.4.1  Effect of Acute BPA Exposure on Neuron Response to Excitotoxic and Oxidative Stress Challenge  When DIV9 and 15 primary rat cortical neurons were pretreated with 10pM (2.283pg/mL) BPA for 1-9h, then challenged with excitotoxic or oxidative stress, there was no significant change in neuronal viability after allowed recovery time as compared to the vehicle controls which also underwent excitotoxic or oxidative challenge (Figures 3.1 and 3.2). The excitotoxic stress protocol used was more effective in the DIV15 neurons (Figure 3.2), resulting in an approximate 40% decrease in cell viability, while the same protocol in DIV9 neurons only resulted in an approximate 20% decrease in cell viability (Figure 3.1). The oxidative stress protocol used only decreased cell viability by approximately 20% for both DIV9 and 15 neurons under all treatment conditions tested.  3.4.2  Effect of Chronic BPA Exposure on Neuron Response to Excitotoxic and Oxidative Stress Challenge  The chronic exposure of primary rat cortical neurons to concentrations of BPA ranging from 1mM (0.228mg/mL) to 1fM (0.228fg/mL) from DIV3 to DIV9 or 15 did not significantly change the number of cells that survived an excitotoxic or oxidative stress onslaught (Figures 3.3 and 3.4). The excitotoxic stress protocol used decreased cell viability by approximately 40% in DIV15 neuron cultures (Figure 3.4). The oxidative stress protocol used decreased the viability of cultures only treated with media by approximately 30% in DIV9 neurons (Figure 3.3). Chronic exposure to 10µM (2.283µg/mL)-100fM (22.829fg/mL) concentrations followed by an oxidative stress challenge only decreased neuron viability by approximately 20% but this increase in cell viability is not significantly different compared to control. In DIV15 neurons, oxidative stress only decreased cell viability by approximately 5%, which is not significantly different from cultures not challenged with oxidative 41  stress (Figure 3.4). Cultures chronically treated with 100µM (22.829µg/mL), 10µM (2.283µg/mL) and 1fM (0.228fg/mL) BPA then challenged with oxidative stress did not have a cell viability significantly different from control. However, cultures treated with 100nM (22.829ng/mL)-100fM (22.829fg/mL) concentrations of BPA had decreased overall cell viability after oxidative stress challenge as compared to media control, 100µM (22.829µg/mL) and 10µM (2.283µg/mL) BPA treatments. The cell viability at these concentrations of BPA were also significantly different after excitotoxic stress challenge from concentration-matched BPA only controls.  42  3.5  Discussion  The pretreatment of rat cortical neurons with 10pM (2.283pg/mL) BPA for 1, 3, 5 or 9h neither exacerbated nor attenuated cell death when the neurons were challenged with an oxidative or excitotoxic stress at both DIV9 and DIV15 (Figures 3.1 and 3.2). Although 10pM (2.283pg/mL) BPA did not decrease cell viability under acute (Figures 2.1, 2.2 and 2.3) nor chronic (Figures 2.4 and 2.5) conditions at both DIV9 and 15, we decided to further investigate its potential to predispose neurons to oxidative or excitotoxic stress because of experiments we were simultaneously conducting with SREBP 1 (see chapter 4). From our preliminary results in chapter 4, we hypothesized that increased activation of SREBP 1 may cause neuronal susceptibility to oxidative or excitotoxic stress in a time-dependent manner that had its maximal effect shortly after 5h but neuronal viability did not change after oxidative nor excitotoxic challenge after preincubation with 10pM BPA for 1, 3, 5 or 9h (Figures 3.1 and 3.2) [96]. The sublethal dose of hydrogen peroxide used did not induce cell death to the extent (40-60% decrease in cell viability) normally seen in the literature at both DIV9 and 15 [120, 121, 122, 123]. This may, however, be due to the fact that culture media was not replaced with Neurobasal without B27 prior to the oxidative stress challenge, as was the case in other studies [120, 121, 122]. In our chronic experiments, culture media was not replaced because we did not want to change the BPA-containing conditions our cultures were grown in. While we knew the approximate concentrations of BPA in our cultures, the concentrations could have changed over time due to BPA being taken up in the cell, BPA degradation once within the cell, or evaporation of culture media. In the acute experiments, we did not replace culture media to maintain continuity with our chronic treatment. Additionally, we were concerned that the act of replacing culture media would stress our cells and confound our findings. B27, in addition to a variety of fatty acids, proteins and hormone supplements, also contains the antioxidants catalase, DL-α-tocopherol, retinyl acetate, glutathione and carnitine [124]. Thus, the presence of B27 in culture media during oxidative stress treatment could alleviate the degree of cell death found in our study. However, in support of our results with oxidative stress challenge, Chang et al (2008) reported that doses of hydrogen peroxide as high as 50µM (11.415µg/mL) 43  only induced 5-12% cell death after 24h in cortical neurons in B27-minus-antioxidantsupplemented Neurobasal media [125]. This number is comparable to the maximum of 20% neuronal death found in our study (Figure 3.1). Chang et al (2008) only reported significant cell death after treatment with 100µM hydrogen peroxide, but our treatments of rat cortical neurons with 80µM hydrogen peroxide also failed to produce significant cell death after 12h (data not shown). In addition, differences in neuron types could be a possible explanation for the discrepancy. Zhong and Lee (2007) conducted their experiments on mouse cerebellar granule neurons, and Guo (2009), Jia (2009) and Chang et al (2008) conducted their studies on mouse cortical neurons, while our experiments were conducted in rat cortical neurons. While our excitotoxic stress challenge decreased cell viability by 40% in DIV15 neurons, it only decreased cell viability by approximately 20% in DIV9 neurons (Figures 3.1 and 3.2). The degree of cell death in DIV15 neurons is comparable to the degree of cell death usually found in excitotoxic stress protocols [96, 121, 122]. The failure of our excitotoxic stress challenge to cause cell death in DIV9 neurons is probably due to the relative immaturity of DIV9 neurons in culture as compared to DIV15 neurons [126, 127, 128]. In rat cortical neurons grown in culture, an increase in NMDA receptor subunit 1 mRNA begins at DIV1, and continues through to DIV21, while NMDA receptor subunit 2B mRNA increases after DIV1 to reach 90% of maximal expression by DIV7, remaining elevated through to DIV21 [129, 110, 130]. NMDA receptor subunit 2A, on the other hand, is expressed at low levels from DIV1-7 and only begins to increase in abundance shortly before DIV14 to DIV21 [129, 110, 130]. Western blots of NMDA receptor subunit 1 show maximal protein expression at DIV14, while NMDA receptor subunit 2B shows near maximal expression at DIV7 and NMDA receptor subunit 2A only begins to be consistently expressed at DIV14 [110]. These changes expression levels of NMDA receptor subunit 2A and 2B in vitro are comparable to developmental stages of maturation in vivo, and may explain our failure to induce excitotoxic stress in vitro [110, 130, 131]. Under chronic BPA treatment, neurons challenged with oxidative stress at DIV9 did not have better or worse survival than neurons treated in parallel without BPA (Figure 3.3). At DIV9, there appears to be some neuroprotection with 44  chronic 10µM (2.283µg/mL) to 100fM (22.829fg/mL) BPA treatment, which may be due to slight antioxidant properties inherent in BPA’s phenolic structure, but the slight increases in light absorbance at 570nm were not significant compared to control (Figure 3.3) [114]. However, hydrogen peroxide treatment did not consistently decrease the same percentage of neurons surviving per experiment, which may also be explained by the antioxidants supplemented in the culture media [124]. At DIV15, there appears to be a non-monotonic dose response in the survival of cortical neuron cultures chronically treated with BPA followed by oxidative stress (Figure 3.4). While oxidative stress did not decrease cell viability in neuron cultures chronically treated with media control and 100µM (22.829µg/mL), 10µM (2.283µg/mL) and 1fM (0.228fg/mL) BPA compared to concentration-matched cultures in the BPA only group, there was decreased cell viability in cultures chronically treated with 100nM (22.829ng/mL)-1fM (0.228fg/mL) BPA. Cultures treated chronically with 100µM (22.829µg/mL)-1fM (0.228fg/mL) BPA appeared to give a U-shaped curve in cell viability following an oxidative stress challenge. At first, we assumed that this was due to non-randomized placement of treatment conditions on the 24-well plate. However, one replicate where positions of treatment conditions were randomized showed increased or no change in cell viability at 100µM (22.829µg/mL), 10µM (2.283µg/mL), 1nM (0.228ng/mL), 10pM (2.283pg/mL) and 1fM (0.228fg/mL) concentrations of BPA compared to neurons treated with oxidative stress only (Figure 3.4). An additional 20% decrease in cell viability was observed with chronic treatment of 100nM (22.829ng/mL) and 100fM (22.829fg/mL) BPA compared to neurons treated with oxidative stress only. The data from this replicate is not specifically shown but was consolidated with non-randomized data to generate figure 3.4. Furthermore, a similar non-monotonic dose response in neuron viability was not observed for treatment groups under the BPA only condition, where the placement of treatment groups were matched to those later challenged with oxidative stress. Therefore, chronic exposure of neuron cultures to 100nM (22.829ng/mL)-10pM (2.2829pg/mL) concentrations of BPA may predispose these neurons to mild oxidative stress that would not normally cause a significant decrease in cell viability. No hormesis was observed in DIV15 neurons chronically treated with BPA followed by excitotoxic stress. DIV9 neurons undergoing chronic BPA treatment were not challenged with 45  excitotoxic stress because previous experiments at DIV9 had already demonstrated that these neurons were too immature to respond appropriately to NMDA-induced excitotoxicity (Figure 3.1). An approximate 35% cell death resulted from the excitotoxic stress challenge to DIV15 neurons undergoing chronic BPA treatment (Figure 3.4). There appears to be slight variations in neuronal survival at different concentrations of BPA, but this is again most likely due to non-randomized placement of treatment conditions in the majority of the experiments, as one experiment with randomized placement did not conform to the same trend (data is not specifically shown but was consolidated with non-randomized data to generate figure 3.4). Neurons that survived 1mM (0.228mg/mL) and 500µM BPA (0.114mg/mL) treatment from DIV3-15 were not susceptible to further excitotoxic or oxidative stressinduced cell death (Figure 3.4). This may be partly because the surviving cells in culture are not pure neurons, but consist of astrocytes or other cell types that do not suffer excitotoxic cell death due to a lack of NMDA receptors.  46  Figure 3.1: Effect of acute BPA pre-treatment on DIV9 cortical neuron response to excitotoxic and oxidative stress. e18 primary Wistar rat cortical neurons were plated in 24-well tissue culture plates at 1.25x105 cells per well. At DIV9, BPA was added to each well to a final concentration of 10pM (2.283pg/mL) at timepoints 0, 4, 6 and 8h to yield total incubation times of 9, 5, 3 and 1h respectively. At 9h, NMDA and glycine or hydrogen peroxide were added into the wells at 50µM and 20µM or 40µM respectively to simulate excitotoxic or oxidative stress. 1h post-NMDA and glycine addition, 100 50µL MTT solution was added to each well after the recovery period (24h for excitotoxic stress, 12h for oxidative stress) and the cells were incubated for a further 24h. Precipitated formazon was dissolved in a solution of 10% glycine in DMSO and the absorbance of each sample at 570nm was recorded in a spectrophotometer. Cell viability is expressed as relative absorbance compared to control (Vehicle). Treatment effects were analyzed with one-way ANOVA. Data for condition BPA only were compiled from figure 2.2. The results for conditions Excitotoxic Stress and Oxidative Stress are presented as means ± SEMs of 3 independent experiments; N=3 for all treatments shown; p <0.05. No statistical significance from the media control was found for either stress challenge.  47  Figure 3.2: Effect of acute BPA pre-treatment on DIV15 cortical neuron response to excitotoxic and oxidative stress. e18 primary Wistar rat cortical neurons were plated in 24-well tissue culture plates at 1.25x105 cells per well. At DIV15, BPA was added to each well to a final concentration of 10pM (2.283pg/mL) at timepoints 0, 4, 6 and 8h to yield total incubation times of 9, 5, 3 and 1h respectively. At 9h, NMDA and glycine or hydrogen peroxide were added into the wells at 50µM and 20µM or 40µM respectively to simulate excitotoxic or oxidative stress. 1h post-NMDA and glycine addition, 100 50µL MTT solution was added to each well after the recovery period (24h for excitotoxic stress, 12h for oxidative stress) and the cells were incubated for a further 24h. Precipitated formazon was dissolved in a solution of 10% glycine in DMSO and the absorbance of each sample at 570nm was recorded in a spectrophotometer. Cell viability is expressed as relative absorbance compared to control (Vehicle). Treatment effects were analyzed with one-way ANOVA. Data for condition BPA only were compiled from figure 2.3. The results for conditions Excitotoxic Stress and Oxidative Stress are presented as means ± SEMs of 4 independent experiments; N=4 for all treatments shown; p <0.05. No statistical significance from the media control was found for either stress challenge.  48  Figure 3.3: Effect of chronic BPA pre-treatment on DIV9 cortical neuron response to excitotoxic and oxidative stress. e18 primary Wistar rat cortical neurons were plated in 24-well tissue culture plates at 1.25x105 cells per well. At DIV3, 100% of the culture media was replaced with fresh media supplemented with concentrations of BPA ranging from 10µM (2.283µg/mL)1fM (0.228fg/mL). At DIV9, hydrogen peroxide was added into the wells to a final concentration of 40µM for 12h to simulate oxidative stress. 50µL MTT solution was added to each well and the cells were incubated for a further 24h. Precipitated formazon was dissolved in a solution of 10% glycine in DMSO and the absorbance of each sample at 570nm was recorded in a spectrophotometer. Cell viability is expressed as relative absorbance at 570nm compared to control (media). Treatment effects were analyzed with one-way ANOVA. Data for condition BPA only were compiled from figure 2.4. The results for conditions Oxidative Stress are presented as means ± SEMs of 3 independent experiments; N=(from left to right) 5, 6, 6, 6, 6, 6, 6; p <0.05. No statistical significance from the media control was found.  49  Figure 3.4: Effect of chronic BPA pre-treatment on DIV15 cortical neuron response to excitotoxic and oxidative stress. e18 primary Wistar rat cortical neurons were plated in 24-well tissue culture plates at 1.25x105 cells per well. At DIV3, 100% of the culture media was replaced with fresh media supplemented with concentrations of BPA ranging from 10µM (2.283µg/mL)1fM (0.228fg/mL). Half of the culture media was replaced with fresh media supplemented with BPA twice a week. At DIV15, NMDA and glycine or hydrogen peroxide were added into the wells at 50µM and 20µM or 40µM respectively to simulate excitotoxic or oxidative stress. 1h post-NMDA and glycine addition, 100% of the media was replaced with fresh media and the cells were allowed to recover for 24h. Cells treated with oxidative stress were incubated with 40µM hydrogen peroxide for 12h. 50µL MTT solution was added to each well and the cells were incubated for a further 24h. Precipitated formazon was dissolved in a solution of 10% glycine in DMSO and the absorbance of each sample at 570nm was recorded in a spectrophotometer. Cell viability is expressed as relative absorbance compared to control (media). Treatment effects were analyzed with one-way ANOVA, followed by the post-hoc Tukey HSD test. Data for condition BPA only were compiled from figure 2.5. The results for conditions Excitotoxic Stress and Oxidative Stress are presented as means ± SEMs of 6 independent experiments; N=(from left to right, beginning with Excitotoxic Stress: 9, 3, 3, 3, 16, 16, 16, 16, 16, 11; Oxidative Stress: 6, 2, 2, 2, 12, 12, 12, 12, 12, 8; p <0.05 (* indicates statistical significance from control).  50  Chapter 4 Effects of BPA on Cell Regulation Mechanisms in Cortical Neurons 4.1  Introduction  In chapters 2 and 3 we have shown that a 5h exposure to 1mM (0.228mg/mL) BPA will cause a 25% decrease in neuron viability. Chronic exposure to 1mM (0.228mg/mL) and 500µM (0.114mg/mL) BPA from DIV3-15 resulted in cell death in 90% of the neuron culture (Figure 2.5). While concentrations of BPA below 100µM (22.829µg/mL) do not cause neuronal cell death, nor predispose neurons to oxidative or excitotoxic stress in our study, low BPA concentrations have been reported to result in morphological changes, cell proliferation, increases in intracellular calcium and neurotoxicity in a variety of different cell lines [5, 7, 9, 58, 59, 60, 61]. A chemical with deleterious effects on the brain does not necessarily have neuronal death as a mechanism of action. Because neurons in the brain form extensive networks with one another, a chemical can produce unwanted effects in behavior simply by changing the ability of neurons to signal through these connections. Examples of this can be found in studies where low doses of BPA changed maternal or sexually dimorphic behavior without causing detectable neuron death [21, 64, 65, 74, 76]. Therefore, it is possible that BPA influences the central nervous system, particularly during development, by subtly modifying the way neurons connect and their ability to intertalk with one another. One possible mechanism through which this might oc-  51  cur is by changes in cell signaling, through either enzyme and/or transcription factor activation.  4.1.1  Sterol Regulatory Element Binding Protein 1  SREBP 1 is a transcription factor normally sequestered in the endoplasmic reticulum. It is cleaved under low sterol conditions, whereupon it translocates to the nucleus to upregulate the transcription of genes involved in fatty acid, phospholipid, cholesterol and triglyceride biosynthesis [94, 95, 96]. In addition to biosynthesis, SREBP 1 also modulates the expression of G-proteins and voltage gated ion channels [97, 98, 99]. Phospholipid and cholesterol content determine the fluidity of the plasma membrane, which influences the signaling and conductance properties of neurons. The density of voltage gated ion channels and their placement in a cell also affects the ability of a neuron to transmit electrical information to its synaptic terminals. In light of these considerations, and the recent discovery that SREBP 1 plays a role in NMDA-induced excitotoxic neuronal death, we decided to assay our rat cortical neuron cultures for changes in SREBP 1 activation after exposure to various concentrations of BPA [96].  4.1.2  Aims of Chapter  In this chapter, we examine the effects of different concentrations of BPA on the activation of SREBP 1 in primary rat cortical neuron cultures after 5h of exposure time. We hypothesize that sublethal concentrations of BPA will cause a measurable increase in the activation of SREBP 1 in our culture system.  52  4.2 4.2.1  Materials and Methods Primary Rat Neuron Culture and Maintenance  See section 2.2.2. Modifications were as follows: primary rat cortical neurons were plated to a density of 7.5x105 cells per well in a 6-well tissue culture plate.  4.2.2  BPA Treatment  See section 2.2.3.  4.2.3  Sample Preparation  At the conclusion of each treatment protocol, the neuron cultures were washed twice with PBS at room temperature. 1 tablet of Complete Protease Inhibitor Cocktail (Hoffmann-La Roche Ltd, Indianapolis, IN) was dissolved in 1mL PBS to make a 50x stock solution. This was diluted to 1x in sample buffer consisting of 1% (w/v) sodium dodecyl sulfate (SDS, Bio-Rad, Hercules, CA) in PBS. After aspiration of culture media, 400µL of this buffer was added to each well. The cells were scraped from the plate with an inverted 1mL pipette tip and boiled at 100◦ Celcius for 5min. The harvested cells were allowed to cool to room temperature, passed six times through a 10µL pipette tip and allowed to sit on the bench for 20min-overnight. Following centrifugation, the supernatant was stored at -80◦ Celcius and any remaining pellet was discarded. Sample buffer (4x) was made from bromophenol blue (Sigma-Aldrich, Saint Louis, MO), glycerol (Sigma-Aldrich, Saint Louis, MO) Tris-HCl (Fisher Scientific, PA) and SDS (Bio-Rad, Hercules, CA). β-mercaptoethanol (Sigma-Aldrich, Saint Louis, MO) was added to the 4x sample buffer at a 1:5 volume ratio. This solution was mixed with protein samples to obtain a 1x sample solution.  53  4.2.4  Protein Quantification  Total protein content of samples were determined with a DC Protein Assay kit purchased from Bio-Rad (Hercules, CA). Bovine serum albumin (BSA, Sigma-Aldrich, Saint Louis, MO) was used to construct a standard curve. After preparation of the assay, the absorbance was read at 750nm.  4.2.5  Western Blotting  Western blots were run on 8% and 12% polyacrylamide gels when visualizing SREBP 1 and Caspase 3, respectively. The gels were made from 30% acrylamide/bis (BioRad, Hercules, CA), 10% SDS (Bio-Rad, Hercules, CA), 10% aluminum persulfate (Bio-Rad, Hercules, CA), and TEMED (Invitrogen, Carlsbad, CA) solutions. 1.5M Tris (Fisher Scientific, PA) at pH8.8 and 1.0M Tris (Fisher Scientific, PA) at pH6.8 were used to make the separation and stacking gels respectively. Running buffer (10x) was made with tris-base (Fisher Scientific, PA), glycine and SDS (Bio-Rad, Hercules, CA), adjusted to pH 8.3 and diluted down to 1x with distilled water prior to usage. Gels were run at a constant 100 volts (V). Transfer buffer (10x) was made with tris-base (Fisher Scientific, PA) and glycine. Prior to usage, this 10x buffer solution was diluted down to a 1x solution consisting of 20% methanol in distilled water. Gels were transferred onto nitrocellulose membranes (Bio-Rad, Hercules, CA) at 100V for 60min when blotting against SREBP 1, and transferred at 100V for 30min when blotting against Caspase 3. β-actin was easily visualized under both conditions. After transfer, nitrocellulose membranes were stained with Ponceau S Solution (Sigma-Aldrich, Saint Louis, MO) to ensure successful transfer of proteins and washed 3x with a tris-buffered saline Tween-20 solution (TBST) composed of Tris, sodium chloride (Fisher Scientific, PA) and Tween-20 (Amersham Biosciences, Sweden). The membranes were then blocked for 1h at room temperature with a 10% milk (Bio-Rad, Hercules, CA) in TBST solution and washed 3x with TBST. Membranes were blotted with SREBP 1 antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, Cat #8984) diluted 1:200 in TBST for 1h at room temperature or overnight at 4◦ Celcius. Activated SREBP 1 gave a band at 65kDa. 54  Membranes were blotted with β-actin antibody (Cell Signaling Technology, Danvers, MA, Cat #4967) diluted 1:1000 in a 3% bovine serum albumin-TBST solution (BSAT) overnight at 4◦ Celcius. A β-actin band was seen at 45kDa. Membranes were blotted with Caspase 3 antibody (AbCAM, Cambridge, MA, Cat #13848) diluted 1:1000 in a 3% BSAT solution overnight at 4◦ Celcius. An activated caspase 3 band was seen at 15kDa, while inactivated caspase 3 showed a band at 30kDa. After primary antibody blotting, membranes were washed 3x with TBST over 1h before incubation with horse radish peroxidase-linked (HRP) secondary antibodies (PerkinElmer) diluted 1:1000 in TBST at room temperature for 1h. Following secondary staining, membranes were washed 3-5x with TBST over 1h before exposure with a Plus-Enhanced Chemiluminescence (Plus-ECL) kit (PerkinElmer, Waltham, MA).  4.2.6  Statistical Analysis  Densitometric analysis of Western Blots were carried out with the program Image J. Statistical analyses were then performed using Instat+ software (Statistical Services Centre, The University of Reading, United Kingdom). Treatment effects were analyzed with one-way analysis of variance (ANOVA). Significance was set at at p <0.05.  55  4.3  Study Design  Effect of various concentrations of BPA on activated SREBP 1 after 5h Primary rat cortical neurons were plated in 6-well tissue culture plates. At DIV1416, diluted BPA was added to each well to final concentrations of 10µM (2.283µg/mL), 1µM (0.228µg/mL), 100nM (22.829ng/mL), 10nM (2.283ng/mL), 1nM (0.228ng/mL), 100pM (22.829pg/mL), 10pM (2.283pg/mL), 1pM (0.228pg/mL0, 100fM (22.829fg/mL), 10fM (2.283fg/mL0 and 1fM (0.228fg/mL). 5h post-BPA addition, the cultures were harvested and prepared for Western Blot analysis.  56  4.4 4.4.1  Results Effect of Various Concentrations of BPA on Activated SREBP 1 after 5h  Primary rat cortical neurons were treated with concentrations of BPA ranging from 10µM (2.283µg/mL)to 1fM (0.228fg/mL) for 5h, then collected for Western Blot analysis to determine if BPA had the potential to change cell regulatory mechanisms. Although there was a slight increase in activated SREBP 1 at 1pM (0.228pg/mL), the difference was not statistically significant from the control (Figures 4.1 and 4.2).  57  4.5  Discussion  The treatment of rat cortical neurons with various concentrations of BPA for 5h resulted in a slight increase in SREBP 1 activation at 1pM (0.228pg/mL) BPA that was not statistically significant from control (Figures 4.1 and 4.2). Although the difference in SREBP 1 activation was not significant at 1pM (0.228pg/mL) BPA, we decided to use a similar concentration (10pM, 2.283pg/mL) for the oxidative and excitotoxic stress challenge experiments conducted on DIV9 and 15 cortical neuron cultures after 1-9h of BPA exposure in chapter 3 (Figures 3.1 and 3.2).  58  Figure 4.1: Effect of 5h BPA treatment on SREBP 1 expression.  Figure 4.2: Quantified effect of 5h BPA treatment on SREBP 1 expression. e18 primary Wistar rat cortical neurons were plated in 6-well tissue culture plates at 7.5x105 cells per well. At DIV15, BPA was added to each well to final concentrations of 10µM (2.283µg/mL)-1fM (0.228fg/mL). 5h post-BPA addition, the cultures were harvested and prepared for Western Blot analysis. The transferred membranes were immunoblotted with antibodies against SREBP 1 and β-actin. SREBP 1 activation is expressed relative to control (NB) and βactin (internal control). Western Blots were analyzed with the program Image J and treatment effects were then analyzed with one-way ANOVA. The results are presented as means ± SEMs of 7 independent experiments; N=(beginning from left to right) 7, 5, 1, 4, 4, 5, 2, 7, 2, 4, 4, 3; p <0.05. No statistical significance from control was found.  59  Chapter 5 Conclusions and Further Studies As described in the previous chapters, the effects of exposure to various concentrations of BPA were studied. Specifically, we investigated the effects of BPA on the viability of neuron cultures, in isolation (chapter 2) and coupled with oxidative or excitotoxic stress (chapter 3). In addition to establishing a concentration-dependent curve of neuron viability under these conditions, we also looked at the time-dependence of neuron viability in cultures treated with 10pM BPA. This concentration of BPA was chosen for its effects on SREBP 1 activation (examined in chapter 4). The results of these investigations are summarized below: 1. The viability of DIV15 primary rat cortical neuron cultures is decreased after 5h exposure to 1mM (0.228mg/mL), but not 500µM (0.114mg/mL)-10fM (2.283fg/mL), BPA. 2. The viability of DIV9 and 15 primary rat cortical neuron cultures is not changed after 1, 3, 5 or 9h exposure to 10pM (2.283pg/mL) BPA. 3. The viability of DIV9 primary rat cortical neuron cultures is not changed after 5h exposure to 10µM (2.283µg/mL)-100fM (22.829fg/mL) BPA. 4. The viability of DIV15 primary rat cortical neuron cultures is decreased after chronic exposure to 1mM (0.228mg/mL) and 500µM (0.114mg/mL), but not 100µM (2.283µg/mL)-1fM (0.228fg/mL), BPA. 5. The viability of DIV9 and 15 primary rat cortical neuron cultures is not changed  60  after 1, 3, 5 or 9h exposure to 10pM (2.283pg/mL) BPA followed by an oxidative or excitotoxic stress challenge. 6. The viability of DIV9 primary rat cortical neuron cultures is not changed after chronic exposure (beginning at DIV3) to 10µM (2.283µg/mL)-100fM (22.829fg/mL) BPA followed by an oxidative stress challenge. 7. The viability of DIV15 primary rat cortical neuron cultures is not changed after chronic exposure (beginning at DIV3) to 100µM (22.829µg/mL)-1fM (0.228fg/mL) BPA followed by an excitotoxic stress challenge. 8. The viability of DIV15 primary rat cortical neuron cultures is decreased after chronic exposure (beginning at DIV3) to 100nM (22.829ng/mL)-100fM (22.829fg/mL), but not 100µM (22.829µg/mL), 10µM (2.283µg/mL) or 1fM (0.228fg/mL) BPA followed by an oxidative stress challenge. 9. The activation of SREBP 1 in DIV15 primary rat cortical neuron cultures is neither increased nor decreased after 5h exposure to 10µM (2.283µg/mL)-1fM (0.228fg/mL) BPA. In conclusion, while environmentally relevant concentrations of BPA may not cause decreased cell viability, they may predispose neurons to mild oxidative stress under chronic BPA exposure (refer to chapter 3). The viability of chronically exposed neurons were decreased only with concentrations of BPA ranging from 100nM (22.829ng/mL)-100fM (22.829fg/mL), but not at 100µM (22.829µg/mL), 10µM (2.283µg/mL) or 1fM (0.228fg/mL). This concentration response curve is U-shaped and may be an example of a non-monotonic dose response, also known as hormesis, of BPA in primary rat cortical neuron cultures. The activation of SREBP 1 in rat neuron cultures after 5h of BPA exposure is slightly greater at 1pM (0.228pg/mL) BPA, but this increase is not statistically significant from control. SREBP 1 is a transcription factor regulating a variety of genes involved in lipid biosynthesis, as well as G proteins and voltage gated ion channels. As a result, changes in activation status in response to acute BPA exposure may have subtle, but deleterious, neurological effects in humans and laboratory animals.  61  Our findings on the neurotoxicity of low molar concentrations of BPA is consistent with other experiments in the literature on cortical neurons as well as other neuronal types. In addition, high concentrations of BPA induced severe neuronal death, supporting other reports in the literature on hippocampal, cerebellar and hypoathalamic neurons and suggesting that neurotoxic concentrations of BPA may be consistent across different neuronal types [56, 7, 59, 58, 63, 108].While there have been reports on potential biochemical mechanisms of action of BPA inside the cell, to our knowledge, none have looked at the effects of BPA on SREBP 1 activation in neurons, nor have they investigated the influence of low and chronic BPA exposure on the ability of neurons to survive neurotoxic stress. As our studies were conducted in vitro, the concentration of BPA exposure was known and accurate at time of administration. This known accuracy will be of use in determining the exposure profile of BPA in future studies. The mechanism behind a non-monotonic dose response to BPA, if it is indeed hormesis, was not investigated in our study. The decrease in cell viability observed after exposure to moderate concentrations of BPA and mild oxidative stress could be due to the idiosyncracies of receptors and ligand-binding. In a commentary on hormesis, Kaiser suggests that receptor-based hormesis could be due to two receptors with different binding affinities to the same ligand [132]. BPA has been reported to bind with low affinity to several different receptors [8, 9, 10, 11]. The contribution of these receptors to the non-monotonic responses we noted in chapters 3 and 4 could be further investigated in both primary cultures and cell lines. In retrospect, the lack of a positive estrogenic control in our studies was an oversight. In our initial studies, the lack of a positive control was not overly deleterious to our results due to the strong replicatability of our data. However, in chapter 4, the lack of a positive estrogenic control made it difficult to determine the mechanism behind activation of SREBP 1 and impeded our efforts to troubleshoot the experiment when it began to fail. We also did not control for BPA contamination of our pregnant rats through their polycarbonate housing cages, nor variations in phytoestrogen exposure in their feed, which could have influenced our results over the course of several months [64].  62  In addition, we did not look at the effects of a strong oxidative stress on neuron cultures chronically exposed to BPA. While we found an interesting, deleterious nonmonotonic response to mild oxidative stress at moderate concentrations of BPA, we should have followed these studies with a stronger oxidative stress challenge. It is possible that this hormetic effect could become more pronounced with a stronger oxidative challenge, but it is also conceivable that the hormetic effect may disappear. This has many implications for humans as well as laboratory studies on animal models. The obvious concerns raised by studies of BPA on laboratory animals relate to human health. However, it is also important to note that BPA contamination could affect and skew our experimental findings at the in vitro and in vivo levels. Indeed, this was how BPA was first identified - in 1993 as a competitor in estrogen receptor binding and in 2003 as an environmental contaminant with adverse effects on female mice reproduction [133, 134]. While BPA is an endocrine-disrupting chemical, its effects on human health and laboratory animals are not limited to reproduction. In vivo studies have shown that at low doses, BPA has a myraid of effects on the laboratory animal, ranging from changes in exploratory, investigative and sexually dimorphic behavior to changes in anatomy, morphology and behavior of neurons in the brain [62, 64, 65, 135]. In addition, evidence is emerging of how developmental exposure to BPA can result in differences in adult behavior [21]. For example, exposure to BPA in fetal life or during gestation changed maternal behavior, but exposure during both periods did not change maternal grooming of pups [21, 64, 74]. This is particularly alarming as it highlights permanent changes in adulthood caused by exposure during development. These changes could be due to the confounding effects of BPA on estrogen-regulated receptor expression in the brain [136]. Most studies investigating the effects of BPA on the brain have focussed on a behavioral endpoint, or on morphological and connectivity changes in known sexually dimorphic areas, such as the hypothalamus and rostral periventricular preoptic areas [65, 108]. However, the effects of BPA on other brain regions, such as those involved in learning and memory, or those that deteriorate first in neurodegenerative disorders, should also be investigated.  63  In hindsight, our investigations were of a crude nature, as we relied primarily on measures of cell death to distinguish an effect. Cell death is the final measure of toxicity. Many negative changes can occur within neurons before a decrease in cell viability can be measured, as illustrated by many in vivo studies [21, 62, 64, 65, 74, 135]. In chapter 4, we reported changes in activation of the transcription factor SREBP 1. The role of BPA in activating transcription factors also deserves further investigation, as transcription factors have wide-ranging effects on the whole function of a cell. In addition to cell signaling pathways and modulation of gene transcription, the effects of BPA on synaptogenesis and synapse maturation should also be examined, as this has developmental, learning and memory implications for humans. Studies have already reported that BPA inhibits sex-specific androgen-induced synaptogenesis in both male and female rats in the hippocampus, as well as female African green monkeys [62, 137, 63, 135, 138]. However, only a few reports looked at the action of BPA alone in neurons [63]. In these studies, it was found that BPA also changed the expression of NMDA receptors in the hippocampus. Further studies should be conducted to elucidate the specific changes BPA produces in the brain. Furthermore, most investigations on BPA and the brain have focussed on effects on neurons. 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