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Estratriene Neuroprotection through Antioxidant, Non-Estrogen Receptor Mediated Mechanisms

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Title:
Estratriene Neuroprotection through Antioxidant, Non-Estrogen Receptor Mediated Mechanisms
Creator:
PEREZ, EVELYN JECIEL ( Author, Primary )
Copyright Date:
2004

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Subjects / Keywords:
Antioxidants ( jstor )
Brain ( jstor )
Estrogen receptors ( jstor )
Estrogens ( jstor )
Lipids ( jstor )
Neurons ( jstor )
Rats ( jstor )
Reactive oxygen species ( jstor )
Steroids ( jstor )
Toxicity ( jstor )

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University of Florida
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University of Florida
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Copyright Evelyn Jeciel Perez. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
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11/30/2004

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ESTRATRIENE NEUROPROTECTION THROUGH ANTIOXIDANT, NONESTROGEN RECEPTOR MEDIATED MECHANISMS By EVELYN JECIEL PEREZ A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2004

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Copyright 2004 by EVELYN JECIEL PEREZ

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For Mom and Dad.

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ACKNOWLEDGMENTS I owe my utmost thanks, gratitude, and respect to my outstanding mentor, Dr. James W. Simpkins. I thank him for his gentle guidance, encouragement, superfluous praise, generosity, and ever-present, reassuring smile. He has a way of making those around him feel important, needed, and welcomed. I feel honored to be a part of his wonderful and nurturing laboratory. I would like to extend my gratitude to my co-mentor, Dr. Michael Meldrum, for the extra help he so graciously gave. I am also indebted to other members of my committee: Dr. Edwin Meyer, Dr. Maureen Keller-Wood, and Dr. William Millard. I thank them for their kind support, their time, and the knowledge they shared with me. Dr. Yun-Ju He and Dr. Elaine Sumners were wonderful mentors in my rotations in their respective labs and I still remember many of the things they so willingly bestowed unto me. I would like to thank Dr. Robin Martin for taking me under her wings my first year in graduate school without this encouragement I do not think it would have been bearable. I would also like to thank Danielle DeCastro, Dr. Feng Li, Anthony Smith, Dr. Bruce Jung, Amanda Crews, Caren Beck, Dr. Pattie Green, Dr. Kelly Gridley, Steve Messina and Dr. Ming Hu. I thank Stephanie McRae for her friendship, encouragement, laughter, and silliness. Who knew there could be another carbon copy of me out there? I thank Dr. Douglas F. Covey and Dr. Zun Y. Cai for their compounds for which I was able to work on this dissertation. iv

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I thank my ‘Texas” family (Dr. Xiaofei (Sophie) Wang, Dr. Shaohua Yang, Dr. Ran Liu, and Dr. Jian Wang) for their love, guidance, and support. I thank them for making me a part of their lives, for encouraging me to be proud of myself, and for accepting me “as is.” I will remember Jian’s gentle nature, as well as the mantras she tried to instill unto me:” just do it” and “you can’t please everyone.” I am blessed to have another sister (and sometimes mother), Sophie Wang. I thank her for not allowing me to have a bad day. I am lucky our lives crossed as my graduate life is filled with many happy memories with her. Ran Liu and Shaohua Yang are my role models in work and home life. I thank them for their concerns about my well being, whether that pertained to my health, happiness, career, or loneliness. I also thank Yi Wen for his friendship, support, and insights into the important scientific and technological advances. I often feel that I do not deserve the support that these wonderful people bestow to me, but I am thankful for it nonetheless. Although I was initially leery about the move to Texas, I do not regret my decision in the least. I am thankful for the opportunity to meet many new colleagues and friends. I cherish the times I was able to spend with Gulnaz Bachlani, Ritu Shetty, Dr. Nathalie Sumien, Dr. Paromita Das, Priscilla Pang, Dr. Sauymen Sarkar, Nila Patel, and Dr. Marianna Jung. I thank them for their stimulating conversations, laughter, and encouragement. I thank Dr. Meharvan Singh and Dr. Michael Forster for sharing their expertise in their respective fields. I thank Kathy Abdella, Kathy Eberst, Milena Palenzuela, Glenda Voorhees, and Debra Castillo for their kindness and generosity. I also extend my gratitude to all faculty, staff and students in the Department of Pharmacodynamics of the University of Florida and the Department of Pharmacology v

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and Neuroscience of the University of North Texas Health Science Center for providing me with wonderful environments in which to gain my graduate career goals. Last, but not least, I would like to thank my wonderful family: Romer, Jasmine, Joy, Jesse, and Ethan. I thank them for their patience, understanding, and love for me. Even though they not quite know what I do for a living, I know they proud of me. I am more proud to be their daughter, sister, and aunt. From them I inherited my work ethic, good nature, propensity to laugh and smile, and generosity. vi

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TABLE OF CONTENTS page ACKNOWLEDGMENTS.................................................................................................iv LIST OF TABLES...............................................................................................................x LIST OF FIGURES...........................................................................................................xi ABSTRACT.....................................................................................................................xiii CHAPTER 1 INTRODUCTION........................................................................................................1 Clinical Studies.............................................................................................................1 In vivo Studies...............................................................................................................2 In Vitro Studies.............................................................................................................4 Mechanisms of Action..................................................................................................4 Estrogens as Antioxidants.............................................................................................7 Estrogens Role in Neurological Disorders...................................................................8 Alzheimer’s Disease..............................................................................................8 Parkinson’s Disease...............................................................................................9 Neuroinflammation................................................................................................9 Aging...................................................................................................................11 Significance................................................................................................................13 2 MATERIALS AND METHODS...............................................................................14 Cell Culture.................................................................................................................14 Culturing of Cell lines.........................................................................................14 Assay Procedures........................................................................................................15 Toxin Exposure...................................................................................................15 Calcein Acetoxymethyl Ether (AM) Assay.........................................................16 GAPDH Activity.................................................................................................17 Measurement of Reactive Oxygen Species.........................................................17 Measurement of ATP Levels...............................................................................17 Lipid Peroxidation Assay....................................................................................18 Protein Assays.....................................................................................................18 Statistics......................................................................................................................19 vii

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3 DEVELOPMENT AND ASSESSMENT OF NEUROPROTECTION ASSAYS....20 Introduction.................................................................................................................20 Materials and Methods...............................................................................................22 Cell Cultures........................................................................................................22 MTT Assay..........................................................................................................22 Alamar Blue Assay..............................................................................................23 Calcein AM Assay...............................................................................................23 Results.........................................................................................................................23 Detecting Relative Cell Number.........................................................................23 Detection of Cell Viability in the Face of Insult.................................................23 Detection of Estradiol Neuroprotection...............................................................24 Varying Cell Culture Conditions.........................................................................25 Discussion...................................................................................................................25 4 STRUCTURE-ACTIVITY RELATIONSHIP OF ESTRATRIENES.......................39 Introduction.................................................................................................................39 Materials and Methods...............................................................................................39 Cell culture..........................................................................................................39 Cell Viability.......................................................................................................40 Data Analysis and Statistics................................................................................40 Results.........................................................................................................................40 Estratriene Protection against Glutamate and IAA Toxicity...............................40 Correlations between Estratriene and Glutamate or IAA Toxicity.....................40 Estradiol and Other Known Estratrienes.............................................................41 A-Ring Derivatives..............................................................................................41 B-and C-Ring Derivatives...................................................................................43 D-ring Derivatives...............................................................................................44 Discussion...................................................................................................................45 5 ANTIOXIDANT CAPACITY OF ESTRATRIENES...............................................54 Introduction.................................................................................................................54 Materials and Methods...............................................................................................55 Cell Culture.........................................................................................................56 DCF Assay...........................................................................................................56 TBARS................................................................................................................56 Statistics...............................................................................................................56 Results.........................................................................................................................56 Effect of Glucose Oxidase on Production of Reactive Oxygen Species.............57 Effect of Estratrienes on Scavenging Reactive Oxygen Species.........................57 Effect of Iron Chlorideinduced Lipid Peroxidation on Homogenates..............57 Effect of Estratrienes on Iron-Induced Lipid Peroxidation.................................58 Correlation Between Inhibition of TBARs and Glutamate and IAA Protection.58 viii

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Discussion...................................................................................................................59 6 ESTROGEN RECEPTOR INVOLVEMENT............................................................68 Introduction.................................................................................................................68 Materials and Methods...............................................................................................69 Cell Culture.........................................................................................................69 Cell Viability.......................................................................................................69 Estrogen Receptor Binding Assay.......................................................................69 Data Reduction....................................................................................................70 Statistical Analysis..............................................................................................70 Results.........................................................................................................................71 ICI 182,780 Toxicity in HT-22 and SK-N-SH cells............................................71 Tamoxifen Toxicity in HT-22 and SK-N-SH cells.............................................71 4-Hydroxytamoxifen Toxicity in HT-22 and SK-N-SH Cells............................71 Antagonists Effect on Cell Insults.......................................................................71 Estrogen Receptor Binding..................................................................................72 Correlation between Inhibition of TBARs and Glutamate and IAA Protection.73 Discussion...................................................................................................................74 7 SUMMARY AND FUTURE DIRECTIONS.............................................................85 APPENDIX A STRUCTURES...........................................................................................................92 B TABLE OF EC 50 (IC 50 ) VALUES...........................................................................101 C SPEARMAN’S RANK CORRELATION...............................................................103 LIST OF REFERENCES.................................................................................................104 BIOGRAPHICAL SKETCH...........................................................................................152 ix

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LIST OF TABLES Table page 4.1 Potencies of A-ring modified compounds...................................................................52 6.1 Table for EC50 values for ER and ER competition binding experiments with known estrogenic compounds..................................................................................79 x

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LIST OF FIGURES Figure page 2.1 Photomicrograph of cell lines used for experiments....................................................15 3.1 Cell density versus assay response.............................................................................30 3.2 HT-22 cells were seeded at either 3,000 or 5,000 cells per well in 96-well plates.....31 3.3 Effect of various insults on HT-22 cells seeded at different densities using three different cell viability assays....................................................................................32 3.4 Effect of E2 on glutamate and IAA-induced cell death..............................................33 3.5 Photomicrograph of HT-22 cells................................................................................34 3.6 Effect of IAA concentration and time on HT-22 cell viability...................................35 3.7 Pretreatment, Cotreatment, and Posttreatment effects of E2 on glutamate-induced cell death..................................................................................................................36 3.8 Antioxidants and reducing agents protect against glutamate and IAA toxicity..........37 3.9 Effect of glucose and serum concentration on glutamate-induced cell death.............38 4.2 Examples of estratriene-mediated neuroprotection against glutamate-induced cell death.........................................................................................................................49 4.3 Examples of estratriene-mediated neuroprotection against IAA-induced cell death..50 4.4 Spearman’s rank correlations between two doses of glutamate and two doses of IAA...........................................................................................................................51 4.5 Spearman’s rank correlations for glutamate and IAA neuroprotection data..............51 4.6 Average EC50 values of A-ring substituted rings......................................................53 5.1 Effect of glucose oxidase and hydrogen peroxide on reactive oxygen species levels as detemined by DCF Assay....................................................................................62 5.2 Effect of estratrienes on ROS.....................................................................................63 xi

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5.3 MDA standard curves assessed (a)fluorometrically and (b) spectrophotometrically.........................................................................................................64 5.4 Effect of iron chloride on homogenates of (A) rat brain, (B) HT-22 cells, and (C) C6 glioma cells..................................................................................................64 5.5 Estratriene-mediated inhibition of iron-induced lipid peroxidation..........................65 5.6 Correlation between inhibition of iron-induced increases in rat brain homogenates and neuroprotection potency in glutamate and IAA neuroprotection models.........66 5.7. Graphical representation the relationship between lipid peroxidation and neuroprotection.........................................................................................................67 6.1 Effect of estrogen receptor antagonists on HT-22 and SK-N-SH cell viability........77 6.2 Effect of ICI182,780 and tamoxifen on cell toxicity.................................................78 6.3 Effect of estratrienes on estrogen receptor binding...................................................80 6.4 Correlation between estrogen receptor binding and neuroprotection. potencies in glutamate and IAA-induced cell death models....................................................81 6.5 Correlation between estrogen receptor binding and neuroprotection potency in glutamate and IAA cell death models......................................................................82 6.6 Correlation between ER and ER binding and inhibition of iron-induced lipid peroxidation..............................................................................................................83 6.7 Graphical representation the relationship between ER binding, lipid peroxidation and neuroprotection data..........................................................................................84 xii

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Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy ESTRATRIENE NEUROPROTECTION THROUGH ANTIOXIDANT, NON-ESTROGEN RECEPTOR MEDIATED MECHANISMS By Evelyn Jeciel Perez May 2004 Chair: Michael Meldrum Cochair: James W. Simpkins Major Department: Pharmacodynamics Estrogens possess neuroprotective properties. Postmenopausal estrogen-replacement therapy has been shown to prevent or delay the onset of Alzheimer’s disease. In light of the fact that oxidative stress is implicated in many neurodegenerative diseases, it is reasonable to consider that estrogens exert their neuroprotective actions via an antioxidant mechanism. The purpose of this study was to evaluate the structure-activity relationship (SAR) among rationally designed estratrienes and their ability to inhibit oxidative stress-induced toxicity, their ability to inhibit markers of oxidative stress, and their ability to activate classical estrogen receptor/estrogen response element mechanisms. The structure-activity relationship (SAR) was determined using a model based on oxidative-stress cytotoxicities induced by glutamic and iodoacetic acids. We confirmed the role of a phenolic A-ring for neuroprotection and expanded these findings by showing xiii

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that modifications made to the estradiol pharmacophore that increases phenoxy radical stability increases neuroprotective potency. Further, we extended that relationship in a lipid-based antioxidant potential model. We found that these potent antioxidant protectants did not bind to estrogen receptors. Not only was there a lack of correlation between binding and neuroprotection, but there was a slight negative correlation in the affinity of the ligand for the estrogen receptor and its ability to protect against oxidative stress. In conclusion, the antioxidant capacity of estratrienes protects neuronal cells against oxidative stress in a manner that is not mediated by classical estrogen receptor mechanisms but through its ability to prevent lipid oxidative stress events. xiv

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CHAPTER 1 INTRODUCTION Estrogens are steroid hormones that are produced mainly by the ovaries, but are also formed by aromatase P450 conversion of androgens (and other C19 steroids) in the periphery (bones, adipose, and adrenal gland) and central nervous system (CNS). 1 Naturally occuring estrogens include estrone, estradiol, and estriol. 17-estradiol (estra-1,3,5(10)-triene-3,17-diol, E2) is the most potent estrogen receptor-acting estrogen. Depending on the phase of a normal-cycling adult female, the ovarian follicle secretes 70 to 500 g of estradiol daily (Novartis, Vivelle-Dot Drug Insert). After the menopause, estrogen levels drop severely but are still made by conversion of androstenedione by aromatase. They exert a plethora of effects on multiple target tissues including the adult brain. In fact, much research has focused on estrogens as neurotrophic, neuroregenerative, and neuroprotective agents. 2-5 Clinical Studies Women in an estrogen-deprived state are at risk for stroke and neurodegenerative diseases. Epidemiological evidence suggests that post-menopausal estrogen replacement therapy (ERT) reduces the risk or delays the onset of Alzheimer’s disease (AD). 6,7 Estrogen loss from natural or surgical menopause has been associated with a decline in cognitive function 8-10 that was reversed by ERT. ERT has also been shown to affect cognitive function during normal brain aging as well. 11-15 Others find estrogen-amelioration of disease in Parkinson’s disease (PD), 16,17 an animal model of amyotrophic lateral sclerosis (ALS) 18,19 (but not in human populations), 20 and recovery from 1

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2 neurotrauma such as stroke 4,21 in some but not all cases. 22 Mortality from stroke was reduced in post-menopausal subjects who were taking ERT at the time of stroke. 23-25 In vivo Studies In vivo, estrogens are protective against a variety of injury paradigms. Studies have shown that E2 protects basal forebrain cholinergic neurons from NMDA infusion 26 and fimbrial lesion. 27 Following entorhinal cortex (EC) lesion, OVX reduced and ERT increased the growth of commissural-association fibers, 28,29 a response that was linked to apolipoprotein E. 30,31 There are also reports of estrogen’s neuroprotective effects on hippocampal cells after kainate 32,33 and lithium-pilocarpine-induced 34 status epilepticus (SE) in female rats; estrone, but not E2, was able to protect male mice from this insult. 35 E2 protection was linked to GABA inhibition in this kainate-induced SE model system. 36 In kainic acid treated rats, estrogen was not able to affect seizure severity but was able to protect against hippocampal cell loss, seizure-induced damage and reduced the mortality rate. 37 In another kainate-treated rat model, estradiol benzoate was able to delay the onset of clonic seizures but not that of SE, but was also protective in reducing the mortality rate. 32 In an NMDA-induced seizure model, E2-treated OVX animals presented a decrease in the total seizure duration and total seizure number by half when compared to OVX only animals. 38 E2 has recently been shown to prevent cerebellar damage and associated behavioral decline in a rat model of ethanol withdrawal 39-41 as well as a 3-acetylpyridine-induced cerebellar ataxia model. 42 E2 is also able to reduce ischemic lesions in animals subjected to middle cerebral artery occlusion (MCAO); 43-47 estradiol-mediated protection against cerebral ischemia was seen in young, 43,48 middle-aged, 49 and diabetic 50 rats, as well as in mice 51 and gerbils. 52,53 In gerbils, E2 protection was associated with an attenuation in the release of

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3 excitatory amino acids, 46 and E2 protection was seen in male gerbils as well. 54 Other estrogens and nonsteroidal estrogens (17E2, estrone, diethylstilbestrol) were also protective in gerbil model of global ischemia. 55 E2 was also shown to alleviate injury against subarachnoid hemorrhage in rats. 56 In a murine global ischemia model, E2 protection was apolipoprotein E-dependent. 57 The severity of ischemic damage in spontaneously hypertensive rats is dependent upon estrogen status; i.e., rats subjected to MCAO during proestrous periods showed less damage than rats injured during metestrous times. 58,59 Further, injury due to MCAO was decreased in rats whether with pretreatment with physiological levels of E2 47 or with pharmacological post-treatment methods. 45,60 Estrogens also play a role in brain trauma. 3,61 Intact female rats fared better than males against fluid percussion injury as did exogenous E2-treated versus OVX controls. 62 However, estrogen protection was only seen in males. 63 After penetrating brain injury as well as kainic acid-induced injury, 64 an upregulation of aromatase activity was seen in astrocytes. 65 In adult gonadectomized male hamsters, estradiol induced a 30% increase in the rate of facial motor neuron regeneration in an axonal regeneration model. 66 In immature rats, estrogen via an ER-mediated mechanism increased the number of surviving motor neurons by 71% in rats subjected to facial nerve axotomy. 67 Not only does exogenously-applied estradiol decrease the insult-induced toxicities, but the brain, through aromatase activity, upregulates estrogen synthesis at these sites of injury in astrocytes/glia. 68-70 Estrogen receptor (ER) expression is likewise upregulated in response to injury 71,72 and age. 73,74 In the latter case exposure to estrogens with this increase in ER expression was deleterious and in vitro models also show this estrogen

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4 induced toxicity in cell lines that express high levels of estrogen receptors. 75 In aromatase knock-out mice as well as fadrozole (aromatase-inhibitor)-treated intact wild-type mice, ischemic injury is more pronounced compared to wild-type counterparts and E2 replacement is protective. 76 In Vitro Studies In vitro, E2 has been shown to protect cells from a variety of insults. E2 was able to protect neurons from deprivation of serum, 77-80 oxygen-glucose, 81-83 and growth factors. This hormone was also able to defend against the toxicity of various beta amyloid peptides, 77,84-94 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), dopamine (for review see 95-97 ), iron, quinolinic acid 98 and hemoglobin. 81 Further, estrogens ameliorated death from glutamate-induced oxidative stress, 99-101 as well as glutamate, NMDAor kainate-induced excitotoxicity. 102-104 Estrogens inhibited death due to heavy metals (cobalt and mercury) 105 and iron chloride. 106 E2 protected against gp120, the coat protein of HIV, 107,108 HIV regulatory protein (TAT), 109 and exposure to HIV-1 protease. 110 Estrogens have also preserved neuronal cell lines from known apoptosis inducers, such as staurosporine, 111 ethylcholine aziridinium, 112 and thapsigargin. 113 Neuronal extracts collected from primary cultures treated with E2 for as little as 10 minutes have been shown to inhibit caspase-6 activity; E2 was able to inhibit apoptosis in cells injected with this enzyme by induction of a caspase inhibitory factor. 114 Mechanisms of Action The mechanism of estrogen neuroprotection is not yet defined. Estradiol is a pleiotropic compound with many actions that work through genomic and nongenomic pathways.

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5 Classical mechanism for steroids involves genomic actions involving estrogen receptors and estrogen response elements (ERE). ERs belong to the steroid and thyroid hormone receptor subfamily. ER is complexed with heat shock proteins in its unbound state. Once ligand binds, a conformational change occurs which releases HSP and reveals the DNA binding domain. The ligand-ER complex dimerizes with another complex and they bind to an estrogen response element (ERE) to initiate transcription. Protective mechanisms may involve the regulation of genes including growth factors (VEGF, EGF, BDNF, NGF) 115-117 as well as their cognate receptors (trkA; 118 ), immediate early genes, 119-121 Bcl-2, 71,122 Bcl-XL, 123,124 heat shock proteins, 125 CREB, 126 tau, 127 Nip-2, 128 glutamine synthetase, 129 and galanin. 130 Estradiol not only affects an ER/ERE pathway to elicit genomic effects, but it can also affect AP1 actions. ER is a transciptional activator for the AP1 site, while ER is an inhibitor. Further, the estrogen receptor does not have to be bound by estradiol to be activated. Estrogen receptors (ER) are ligand-activated enhancer proteins whose activity is regulated by co-repressors (nuclear receptor co-repressor) and co-activators (caveolin-1). There have been two characterized ERs (ER and ER) 131 that are encoded by two different genes (chromosome 6 for ER and chromosome 14 for ER) and are found in a wide variety of neuronal tissues, 132 although ER and ER have distinct patterns of tissue distribution. While ER is mainly found in reproduction-related tissues (uterus and hypothalamus), ER is more widespread (cortex, hippocampus, cerebellum, olfactory lobe, cardiovascular system, and immune system). These receptors have similar DNA binding domains. ER and ER can homodimerize or heterodimerize. 133,134 Also, another receptor termed ER has been found in teleosts. 135

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6 Of late, much research has focused on membrane estrogen receptors, 136-142 thus compounding the multiplicity of estrogen action. These membrane receptors were derived from the same transcript as that of nuclear estrogen receptors. 143 Estradiol-bound membrane receptors have been shown to reduce calcium currents in rat neostriatum, 144 and to potentiate kainate currents 145 in hippocampal neurons that lack intracellular ER 146 Membrane ER localize in caveolae, which creates an environment for cross-talk with other signaling components, such as those of the MAPK pathway. Caveolins also play a role in ligand-dependent and independent ER signaling. 147 ER, via its AF-1 domain, binds to caveolin-1 to induce estrogen-independent potentiation of ERE-driven transcriptional events. 148,149 Evidence for such membrane receptors was deduced from rapid estrogenic effects. 150,151 Also, inhibiting estradiol entry into cells by conjugation to large, non-diffusible proteins (BSA) does not inhibit the hormone’s effect. 150 Further, the addition of RNA (actinomycin D) and protein (cyclohexamide) synthesis inhibitors does not block estrogen effects. 152 Estrogens have also been shown to interact with neurotransmitter systems, including the noradrenergic, 153-156 dopaminergic, 157 cholinergic, 158-160 glutaminergic, 161 and serotonergic 162 pathways, whether it be by altering ligand-binding, direct receptor actions, or levels of neurotransmitters. Estradiol affects adult cellular brain morphology and has been shown to affect axonal sprouting, 29 neuron regeneration, 66,163 neurogenesis, 164,165 synaptic plasticity, 166-169 and neurite growth. 170 Syntaxin, presynaptic proteins (synaptophysin), and postsynaptic proteins (spinophilin) were increased in the CA1 region of female rhesus monkeys. 171 Furthermore, estrogens affect various signal

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7 transduction pathways. They include the Akt, 82,103,172-176 MAPK, 92,98,150,172,177-180 PKC, 94,100,181 PKA/CREB, 126,182-190 IGF-1, 191-196 and NFB, 197-200 and Ca2+ 144,201-203 signaling pathways. The promiscuous nature of estrogens to affect a variety of signaling pathways makes interpretation of individual pathways difficult as there may be cross-talk between the signaling cascades. 204 It may be that estrogens affect something in general that links these pathways. Estrogens as Antioxidants Estrogens have long been recognized as antioxidants in a variety of in vivo and in vitro models. This is important as many neurodegenerative disorders and brain injuries involve an oxidative stress component. This specificity may be due in part to the richness in polyunsaturated fatty acids of neuronal membranes, which increases lipid susceptibility to oxidative damage. Estrogens, with their radical scavenging activity, 53 are able to inhibit oxidative stress markers such as lipid peroxidation. 106,205,206 In cell free systems, inhibition of iron-induced lipid peroxidation may be due to estrogen’s interaction with iron; estrogen can decrease the oxidation of Fe(II) to Fe(III). 207 Estrogen’s antioxidant activity has been narrowed down to its phenolic A ring component. 78,99 Estrogens may work synergistically with other antioxidants, such as reduced glutathione (GSH), to exert neuroprotective effects. 208,209 The four-ring steroidal structure may play a role in decreasing membrane fluidity 210-212 which aids in inhibiting lipid peroxidation cascade events. Others have suggested that estrogens and/or its metabolites may act as “electrophile/redox active factors” 213 to regulate genes under the control of an antioxidant/electrophile response element.

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8 Estrogens Role in Neurological Disorders Alzheimer’s Disease As oxidative stress has been implicated in neurodegenerative diseases, it is not suprising that estrogens have been implicated in neuroprotection in Alzheimer’s disease (for review see Honjo 214 ). Factors that may be responsible for the pathology of AD include apolipoprotein genotype, tau hyperphosphorylation, and amyloid precursor protein dysmetabolism. Estrogen’s role in AD could be due to alterations in APP processing, 177,215-221 or apoE, 222 ERPPXX, 223 ER and/or ER polymorphisms. 224-226 Cerebrospinal estradiol levels were lower in an AD group versus control, and among the AD group, estradiol levels were inversely correlated with A42 levels. 227 Estradiol’s effect on the cholinergic system may also play a role in ERT protection in this disease as well. Estradiol causes an allosteric potentiation of acetylcholine-induced responses by increasing the open probability time of 42 human neuronal nicotinic receptors. 228 Ovariectomy reduced and ERT increased choline acetyltransferase (ChAT) activity, 160,229-232 and E2 affects levels of high affinity choline uptake (HACU) 158,160 which correlated with the memory-related active avoidance behavior test. 160 Estrogen may decrease glutamate toxicity as it has been shown to upregulate GLAST and GLT1, glutamate transporters, in AD-derived cortical astrocytes. 233 Interestingly, GLAST activity is involved in glucose transport as well, and glucose transport is also impaired in the AD brain. Estrogens are known to affect glucose metabolism. In rat brain, estrogens acutely enhance cerebral glucose utilization, 234 and further, glucose use in the brain fluctuates with endogenous levels during the estrous cycle. 235 In primate cerebral cortical

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9 areas, three days of E2 treatment in OVX animals produced an upregulation in the glucose transporters, GLUT3 and GLUT4. 236 Parkinson’s Disease Estrogens have recently been associated with Parkinson’s disease. 97 ERT has been shown to decrease the development of dementia in PD patients, but did not reduce the risk for PD. 16 In vivo, estrogen potentiates dopamine (DA) synthesis and release, 237,238 and alters basal firing rates 157 in adult rats. DA depletion, on the other hand, by substances such as 6-hydroxydopamine, MPP+, and methamphetamine is toxic to the nigrostriatal dopaminergic pathway and estrogens have been shown to modulate these actions. Estrogens protected against 6-hydroxydopamine toxicity to the striatum. 239 This depletion of DA by 6-hydroxydopamine is more pronounced in intact male and OVX female rats versus intact female rats. 240 Estrogen also affected methamphetamine-induced DA-depletion in striatal tissue and rats. 241 Estrogens also prevent MPP+ actions on DA depletion 242,243 and protect against MPP+ toxicity in in vitro 244-247 ; and in vivo settings. 244,248,249 Phytoestrogens and estrogens decreased MPP+-induced cytoxicity and reversed the decrease in dopamine transporter expression. 246 In a different brain system, E2 inhibited organic cation transporter-mediated uptake of MPP+ and thus MPP+-induced increases in cell toxicity, caspase-3 activity, and TUNEL-positive cells in primary cerebellar granule neurons. 250 Neuroinflammation Although it was once thought to be protected from the immune response (immune-priviledged), it has become clear that the brain is immunologically active. 251-255 Cytokines such as interleukins, tumor necrosis factors, and transforming growth factors have been shown to influence disease states of the brain when present chronically.

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10 Alzheimer’s disease, for example, is reported to have an inflammatory component. For example, interleukins (IL-1, IL-1, IL-6) colocalize to senile plaques. Chronic use of nonsteroidal anti-inflammatory drugs, such as aspirin and ibuprofen, have been shown to reduce the risk of AD. 256,257 In age-related conditions, serum IL-6 levels are negatively correlated to cognitive function in elderly patients. 258 Estrogens have been found to influence neuroinflammatory responses. 259 Estrogens reduced glial fibrillary acidic protein (GFAP) expression in male rats injected with the inflammatory agent, lipopolysaccaride (LPS). 260 They have been shown to modulate the expression of proinflammatory genes, such as those encoding cytokines. 197 Aspirin and E2 have also reduced A42 and LPS-induced increases in NFB activation. 197 In vitro, estradiol significantly enhanced human cortical microglia uptake of fluorescent A; E2 effects were reduced but not abolished by 10 M ICI 182,780 (estrogen receptor antagonist), did not coincide with ER-immunoreactivity, and were not specific for A peptides alone. 261 Low nanomolar concentrations of E2 inhibited LPS-induced increases in superoxide production and phagocytotic activity in the N9 microglial cell line. 262 In this same cell line, higher micromolar concentrations of E2 were able to decrease LPS-induced increases in nitrite, iNOS, and TNF levels. 263 Estradiol outperformed thiol antioxidants and spin trap agents (PBN) in their ability to reduce advanced glycation end product (AGE)-induced increases in nitrite and iNOS levels in microglial cells. 264 However, in another model, chronic E2 and/or LPS has a deleterious effect on female mice and exhibited activated microglia and impairment in a water maze behavioral test. 265 In young, but not reproductively senescent female rats, estradiol was able to decrease NMDA-induced IL-1 increases from activated microglia. 266

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11 In experimental autoimmune encephalomyelitis (EAE), a model for multiple sclerosis, pregnancy has been shown to ameliorate the disease signs. This amelioration was suggested to be due to estrogens. In a more definitive study, the semisynthetic estrogen, ethinyl estradiol, has been shown to protect against EAE-induced proteolipid protein 139-151 peptide. 267 Estrogens have been shown to inhibit the migration of TNF-expressing T-cells and macrophages into the central nervous system, which thus prevents inflammation and demyelination. 268 Aging Estrogens play a role in aging as well. 269 Aging is a process that has been associated with an increase in oxidative stress, whether that is by increased production of reactive oxygen species, decrease in defense against these toxic compounds, or the combination, as well as a decrease in protein turnover. The accumulation of oxidative damage is a major factor in the age-related decline in physiological health. Aging also results in a decrease in glucose utilization. There is an increase in cerebral metabolic activity in menopausal women. 270,271 Compared with older women not on estrogen therapy and older men, positron emission tomography revealed that female on estrogen showed a significant increase in glucose metabolism in the lateral temporal region. 272 In vivo, estradiol also causes an increase in glucose utilization. 234,235,273,274 OVX female rats show significantly lower capability of brain glucose utilization, while estrogen replacement causes a 30% increase in glucose utilization and enhanced GLUT1 mRNA and protein. 273 Also, E2 replacement enhances GLUT1 levels in surrounding cerebral infarcts, an effect that may account, in part, for the reduced ischemic damage in animals receiving estrogen replacement. 274

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12 Cellular energy deficits in aging may be due in part to mitochondrial function decline. For example, mitochondrial enzyme activity, such as cytochrome oxidase and adenine nucleotide translocase activity, are markedly reduced in aged animals and reductions in productivity of these enzymes may be related to oxidative damage. In cell culture systems, estradiol stabilizes mitochondrial function 275,276 and ATP levels (through binding to a subunit of ATP synthase/ATPase). 277 Female middle aged and older mice exhibit an increase in astrocytes and microglia that is not seen in their B6 male mice equivalents. This is important, as activated astrocytes and microglia are mediators for the proinflammatory response (complement, cytokines, TNF, etc). In aged OVX mice, Mac1 (microglia)and GFAP (astrocytes)reactivity was decreased by estrogen administration in the dentate gyrus and CA1 region of the hippocampus; GFAP-staining revealed hypertrophy and an increase in the number of processes. 278 Cognitive decline is another symptom of aging. The cognitive function decline seen with age is affected by estrogens. 279 From twin studies, postmenopausal estrogen replacement protects against the onset of age-related cognitive impairment (Cecilia Magnuson, 8 th International Conference on AD in Stockholm Sweden). E2 replacement also enhances memory and cognition in women who have undergone surgical menopause. 10,280 ERT improves cognitive function in women diagnosed with AD in several clinical trials. 281-284 In aged (22 years7 months) rhesus monkeys, animals that do not develop AD, E2-treated OVX animals outperformed their OVX counterparts on a visuospatial working memory test that is indicative of a functional dorsolateral prefrontal cortex. Further, they performed as well as young (approximately 5.2 years 5 months)

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13 intact monkeys in this test. 285 Estrogen replacement does not always negate declines in cognitive function seen in aging. While estrogen ameliorates cognitive decline in middle-aged OVX animals, the effect in older females is not seen and this discrepancy is more prevalently seen when scopolamine is injected. 286 This age-dependent protective response is also substantiated in animal models of NMDA-induced inflammatory responses. 266 Significance The loss of estrogens after the menopause has been linked to a greater vulnerability to various diseases. With increases in the lifespan, women can spend up to one-third of their lives in a hypoestrogenic state. Estrogen use is not without risk, as HRT raises concerns about increased risk of breast and endometrial cancer in women and feminizing effects in males. The potential clinical benefits of estrogen replacement for enhancing cognitive function may not outweigh the associated central and peripheral risks. There is, therefore, a need to find alternative sources of estrogenic agents that do not elicit the carcinogenic effects of estradiol. The proposed research will investigate the mechanism by which estrogens, as an antioxidant, protects neurons against oxidative stress. Mechanisms that lead to an increase in antioxidant activities would therefore be of benefit. This will lead to promising methods (modalities) of protecting neurons against oxidative stress.

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CHAPTER 2 MATERIALS AND METHODS Cell Culture Culturing of Cell lines SK-N-SH neuroblastoma cells were obtained from American Type Tissue Collection (ATCC; Rockville, MD) and were maintained in RPMI-1640 media supplemented with 10% charcoal-stripped fetal bovine serum (FBS; HyClone, Logan, UT), 20 g/ml gentamycin (Sigma, St. Louis, MO) in monolayers in Nunc 75 cm 2 flasks (Fisher Scientific, Orlando, FL) at standard cell culture conditions (5% CO 2 , 95% air). Medium were changed three times weekly and backcultured at confluence (every 5-7 days). Cells were observed with a phase-contrast microscope (Nikon Diaphot-300). SK-N-SH cells were used in passes 37-50 and plated at a density of 12,000 to 20,000 cells/well in 96-well plates. HT-22 cells, a murine hippocampal cell line that is a subclone of HT4 cells and does not express estrogen receptors, were the generous gift of Dr. David Schubert (Salk Institute, San Diego, CA). C6 rat glioma cells were obtained from ATCC. These cells were maintained as stated for SK-N-SH cells with the exception of using DMEM media. HT-22 cells were used in passes 15-30 and seeded at 3,000 to 5,000 cells/well in 96-well plates. C6 cells were used at passages 38-50 and seeded at ~3500 cells/well in 96-well plates. MCF-7 cells, human breast cancer cells, were obtained from ATCC and were maintained as stated for SK-N-SH cells with the addition of 2.5 g/ml bovine insulin 14

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15 (Life Technologies). Cells were passed at confluence (every 10-14 days). This cell line was used at passages 140-160. a. HT-22 murine hippocampal cells b. C6 rat glial cell c. MCF-7 human breast cancer cells d. SK-N-SH human neuroblastoma cells Figure 2.1 Photomicrograph of cell lines used for experiments. Depicted are (a) HT-22, (b) C6, (c) MCF-7, and (d) SK-N-SH cells. Assay Procedures Toxin Exposure Lyophilized A(25-35) (Bachem,Torrance,CA) was initially dissolved in sterile distilled H 2 O and then to a final stock solution of 1 mg/ml in PBS. This stock was then

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16 diluted to the final concentrations in experimental media. Lyophilized A(1-43) (Bachem) was prepared at a 1 mg/ml stock solution in PBS and incubated at 37C for 4 d prior to use as required for aggregation. 287 Cells were exposed to both A peptides at concentrations ranging from 1 25 M. H 2 O 2 (30% v/v, stabilized, Sigma Chemical Co.) was diluted to a 300 mM concentration in sterile water and then diluted to the final concentration in the appropriate experimental media. Glutamate (Sigma Chemical Co.) was dissolved to a 1 M stock solution in PBS and further diluted in DMEM media. HT-22 cells were exposed to glutamate for ~ 16 hours at 5 mM concentrations. Ferric Chloride (Sigma) was dissolved to a 500 mM stock in dH 2 O. Further dilutions were done in culture media. Glucose Oxidase (GO; ICN) was dissolved in PBS and stored as a stock concentration of 10,000 U/ml. Further dilutions were carried out using culture media to concentrations ranging from 2 mU/ml to 10,000 mU/ml. Iodoacetic Acid (IAA; Sigma) was dissolved in H 2 O at a stock concentration of 100 mM. Further dilutions were carried out using culture media to concentrations ranging from 1 and 1000 M. Calcein Acetoxymethyl Ether (AM) Assay. Calcein AM (Molecular Probes,Eugene, OR) is a nonfluorescent, electrically neutral nonpolar analog of fluorescein diacetate, which passively crosses cell membranes and is cleaved to a fluorescent derivative by nonspecific intracellular esterases . Once cleaved in viable cells, the resultant fluorescent salts are retained by intact cell

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17 membranes. Wells were washed once with PBS and replaced with a 1-2.5 M solution of calcein AM in PBS. Cells were incubated for 15 minutes at room temperature and fluorescence measured using a FL600 Fluorescence plate reader (excitation 485 emission 530). Raw data are represented as relative fluorescence units (RFU). GAPDH Activity The enzymatic activity of glyceraldehydes-3-phosphate dehydrogenase (GAPDH) was measured by a slightly modified method described by Kant and Steck (Methods Enzym, 1974). Cell lysates were collected from HT-22 cells treated with IAA and/or protectant in 100-mm dishes. The reaction was initiated by adding sodium arsenate (0.4 M) and glyceraldehyde-3-phosphate (15 mM, adjusted to pH 7.0). Taking the addition of G-3-P as the starting point of the reaction, absorbance and was measured spectrophotometrically at 340 nm and at 28-30C. Measurement of Reactive Oxygen Species HT-22 cells were seeded into 96-well plates at a concentration of 5,000 cells/well. The nonfluorescent probe 2’, 7’-dichlorofluorescein diacetate (DCFH-DA, Molecular Probes, Eugene, OR) was added at a concentration of 50 M for ~1h under standard cell culture conditions. Some wells did not receive DCF and were treated as blanks. One hour after incubation, wells were washed two times with PBS and replaced with phenol-free media and allowed to adjust for a couple of hours. Treatments were added and plates read periodically with a FL600 Fluorescence plate reader (excitation 485 emission 530). Measurement of ATP Levels Cells were plated in 60mm culture dishes. After 48 h, cells were exposed to various doses of IAA, glutamate, or H2O2 from 15 min to 8 h. Cellular ATP levels were

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18 quantified using a luciferin and luciferase-based assay. Cells were washed with PBS once and lysed with ATP-releasing buffer (100mM potassium phosphate buffer at pH 7.8, 1% Trition X-100, 2 mM EDTA and 1mM DTT). Ten l of the lysate was added to Nunc 96-well plates. ATP concentrations in lysate were quantified using an ATP determination kit according to the manufacturer’s instruction using a standard curve generated by known concentrations of ATP and read using a FL600 luminescence plate reader. Protein concentrations of samples were determined by Bradford assay. ATP levels were calculated as nM of ATP per mg of protein and normalized to levels in untreated control cultures. Lipid Peroxidation Assay Lipid peroxidation was induced with FeCl 3 in rat brain homogenates (RBH). RBH was prepared by sonicating rat brain tissue in 0.9 % NaCl (saline). Test compounds were incubated with 1 mg/ml RBH samples for 30’. Iron chloride was then added at a final concentration of 50 M for 15’ at 45-55C. A solution containing 1% (TBA), HCl, and trichloroacetic acid was then added for a further 1h. Fluoresence was determined at excitation/emission wavelengths of 530/590 nm. Data are represented as relative fluorescence units (RFU) and EC50 values were determined using GraphPad Prism software. Protein Assays Protein concentration was determined by the method of Bradford 288 using bovine serum albumin (BSA) at concentrations ranging from 0.063 to 1 mg/ml as a standard curve.

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19 Statistics All data are presented as mean S.E.M. The significance of differences among groups was determined by one-way analysis of variance (ANOVA) with a Tukey’s multiple-comparisons test for planned comparisons between groups when significance was detected. Tukey’s test is commonly used in biological systems and is the most conservative of the multiple comparison tests. For all tests, P < 0.05 was considered significant. For correlational analysis, EC50 (or IC50) values were ranked and used to determine the effect of parameters with Spearman’s ranked correlations, which measures the monotonic association between variables whose relationship is not linear. The rank-order correlation coefficient, r s , is defined to be the linear correlation coefficient of the ranks.

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CHAPTER 3 DEVELOPMENT AND ASSESSMENT OF NEUROPROTECTION ASSAYS Introduction Estrogens have been shown to protect against various insults in a number of cell lines. The methods by which to assess estratriene neuroprotection are numerous. To test for possible neuroprotective agents, one needs a reliable, robust, sensitive, cost-effective, and safe screening assay. Amyloid beta (A) peptides have been implicated in Alzheimer’s disease pathology in that they comprise a key component of senile plaques in AD and exert toxic oxidative stress-related effects in vitro. 289,290 The physiological A(1-43) peptide as well as the nonphysiological A(25-35) peptide have been used as cytotoxic agents in in vitro AD-like paradigms. A peptides can affect calcium dyshomeostasis, 291 generate intracellular peroxides, induce lipid peroxidation, 86,292 activate microglia, and bind to receptors for advanced glycation end products (RAGE). 293 Glutamate kill can either result from an excitotoxic or oxidative stress-inducing mechanisms. Excitotoxic glutamate paradigms 294,295 are performed acutely using low micromolar concentrations and mimics conditions seen in stroke, epilepsy, HD, and AD (for review see, Lipton) 296 . When using cells lines that lack ionotropic glutamate receptors, glutamate can be added at millimolar quantities for extended periods of time to induce slow oxidative stress conditions. 100 In the latter case, glutamate acts via inhibition of the glutamate/cystine antiporter to subsequently decrease the synthesis of glutathione. 20

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21 Loss of glutathione or glutathione transferase activity is observed in neurodegenerative disorders, including AD and PD. 297-299 Iodoacetic acid (IAA) is a sulfhydryl-binding molecule, which inhibits glycolysis by the inhibition of glyceraldehyde 3-phosphate dehydrogenase (GAPDH). GAPDH is a glycolytic enzyme as such is the oft-used housekeeping protein in the normalization of immunoblot assays. Of late, the oxidative modification of critical cysteines, 300 and subsequent translocation of this oxidized GAPDH has also been shown to cause apoptosis. ATP generated after IAA inhibition of GAPDH is a consequence of oxidative phosphorylation. In animals, IAA has been used to induce striatal excitotoxic lesions 301 and cataracts in rats. 302 In vitro, IAA was also used to model chemically-induced ischemic/hypoxic events in PC12 cells 303 and chick retinal cells 304 when used in conjunction with oxidative phosphorylation inhibitors (sodium cyanide, nitropropionic acid). IAA potentiates the kill induced by transfection of mutant SOD in a mouse motor neuron/neuroblastoma cell line (NSC34) 305 and by A toxicity in rat hippocampus. 306 In a preconditioning paradigm, Guo et.al. 307 found that pretreatment with nanomolar concentrations of IAA protected hippocampal cells from subsequent insults (glutamate, iron, and trophic factor withdrawal). There are a plethora of methods for assessing neuronal viability (for review see Stewart 308 ). They include colorimetric formazan production of vital dyes (MTT, XTS, MTS), Texas Red, fluorescent tags, incorporation assays ([ 3 H]Thymidine uptake,BrdU), dye exclusion (Trypan Blue), manual counting, DNA quantification, flow cytometry, etc. MTT (3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) is one the most common viability assays used. MTT is a yellow dye that is converted to a

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22 blue/purple formazan crystal upon mitochondrial activity, 309 specifically succinate dehydrogenase activity. Succinate dehydrogenase is located in the inner mitochondrial membrane and is a component of the Krebs cycle and the aerobic respiratory chain. Alamar blue, also known as resazurin, is yet another redox indicating dye. This dye is added to culture vessels at 10% of culture medium volume present and is measured by colorimetric or fluorometric methods. It can also be used for endpoint as well as kinetic measurements. The dye is added in the oxidized, blue, nonfluorescent form, and is reduced to a pink fluorescent dye by “cell activity,” such as oxygen consumption through metabolism and its reduction has been shown to be dependent on cellular reducing equivalents. 310 Calcein AM is a nonfluorescent, electrically neutral nonpolar analog of fluorescein diacetate, which passively crosses cell membranes and is cleaved to a fluorescent derivative by nonspecific intracellular esterases. Once cleaved in viable cells, the resultant fluorescent salts are retained by intact cell membranes. Higher relative values are indicative of more live cells. In the present study, we assess each of these methods of determining cell viability against a variety of insults to determine the most appropriate assay for cell viability. Materials and Methods Cell Cultures HT-22 cells were maintained as described in Chapter 2. Cells were seeded in 96-well plates at a density of ~3500 cells per well and allowed to incubate overnight. MTT Assay MTT reduction was determined by incubation with 0.25 mg/ml MTT (Sigma Chemical Co., St. Louis, MO) for 3-4 h at 37C. Crystals were solubilized by overnight

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23 solubilization of the formazan product in 50% N,N-dimethyl formamide, 20% sodium dodecyl sulfate, pH 4.8 or removal of well solution and addition of 100 l of DMSO for 15 minutes. Absorbance values were determined at 590 nm and raw data are represented as optical density (O.D.). Wells containing media without cells were used for background determination in this assay. Alamar Blue Assay Alamar blue was added at a final concentration of 10% (v/v) of the culture medium for 2h before the end of the incubation period, unless otherwise stated. Fluorescence was measured at an excitation and emission wavelength of 530 and 590 nm, respectively. Calcein AM Assay Wells were washed once with PBS and replaced with a 1 M solution of calcein AM in PBS. Cells were incubated for 15 minutes at room temperature and fluorescence measured using a FL600 Fluorescence plate reader (excitation 485 emission 530). Results Detecting Relative Cell Number The MTT, calcein AM, and Alamar Blue assays detected increases in cell number (Fig. 3.1). Linearity in all assay methods was achieved at 24 and 48h when HT-22 cells were seeded between 2,000 and 4,000 cells per well. Detection of Cell Viability in the Face of Insult The MTT assay was more sensitive to the effects of A(25-35 (Fig. 3.2) and detected 20% reduction in live cell numbers at the three doses tested. Cell death from this peptide was detectable when calcein AM was used, but only at the highest concentration of A (Fig. 3.2). IAA (20 M) treatment for 16h was potently toxic in all three assay systems (Fig. 3.3). However, data were misskewed at the lower cell densities

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24 when utilizing the Alamar blue assay and more so with the MTT assay (Fig. 3.3); which may signify a signal-to-noise ratio problem. The calcein AM assay showed IAA-induced kill at all cell densities (16 h incubation) tested and proved toxic to >90% of the HT-22 cells. However, cell death at a seeding density of 16,000 cells per well significantly inhibited toxicity from 20 M IAA by about 10% as compared to the other seeding densities (Fig. 3.3). Glutamate toxicity was highly dependent on cell density. Using the MTT assay, toxicity at all seeding densities did not reveal potent cell toxicity by glutamate. The calcein AM and Alamar blue assays detected this glutamate toxicity in an equivalent manner. At 2000 cells/well, 26.81.7, 18.70.4, and 82.29.1% of cells were resistant to 10 mM glutamate challenge as determined by the calcein AM, Alamar blue, and MTT assays, respectively. Loss of glutamate toxicity when seeding was greater than 4,000 cells/well occurred in all three assays. Detection of Estradiol Neuroprotection Using the calcein AM assay, 16h of glutamate exposure dose-dependently decreased cell viability. E2 (10 M) inhibited cell death by 15.9, 39.4, 40.7, and 40.7% against 5, 10, 20, and 40 mM glutamate, respectively (Fig. 3.4a). Figure 3.5 depicts HT-22 cells treated with 10 mM glutamate and 10 M E2. In the glutamate only group, cells lost their stellate morphology and membrane integrity, as evidenced by the increase in propidium iodide staining. E2 was able to inhibit these changes. IAA exposure for 8h dose-dependently decreased cell viability. E2 (10 M) inhibited cell death by 8.0, 31.3, 38.2, and 20.8% against 10, 20, 40, and 80 M IAA, respectively (Fig. 3.4b). IAA, in a dose and time-dependent manner, affected cell viablity

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25 as assessed by calcein AM when assessed up to 8h (Fig. 3.6a). At 8h, 20 M toxicity was cell-density dependent (Fig. 3.6b); at 16 h it was not (Fig. 3.3a). Varying Cell Culture Conditions Pretreatment for 24h or 5h, followed by a washout, did not protect HT-22 cells from glutamate toxicity (Fig.3.7). Pretreatment for 24h and cotreatment during the time of glutamate challenge did not enhance neuroprotection (data not shown). Estradiol can be given 5h after glutamate administration and still protect (Fig. 3.7). Thiol-based antioxidants and reducing agents, cysteine and dithioerythritol (DTE), respectively, like estrogens, decreases cell toxicity (Fig. 3.8). EC50 values for cysteine neuroprotection against 10 mM glutamate and 40 M IAA were 30.6 and 57.5 M, respectively. DTE produced similar potencies and required 24.8 and 31.6 M concentrations to inhibit glutamate and IAA-induced toxicity by 50%, respectively. Aside from cell density-dependent glutamate toxicity, there was also a dose-dependent effect for glutamate toxicity when serum and glucose levels were varied in HT-22 cells (Fig. 3.9). Discussion The present studies attest to the complicated neuroprotection data seen in the literature. Cell responses to deadly stimuli are dependent on time of assay, cell density, method of viability assessment, choice of insult among many other factors. The reduction of MTT is indicative of cellular metabolic activity. Reduction can take place in the mitochondria as well as the cytoplasm. MTT is reduced by the mitochondrial enzyme, succinate dehydrogenase, and involves the hydride ion donation from pyridine nucleotides. While it is a quick and easy assay to perform, a disadvantage of this assay is the need to kill cells, which inhibits further study. Colorimetric assays, in

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26 general, are less sensitive than fluorescent, chemiluminescent, and radiometric assays and thus limit its use as a cell viability marker. The ability of the calcien AM and Alamar Blue assays to detect cell death and the inability for detection with the MTT assay may signify multiple mechanisms for MTT reduction. Further, its use in detecting E2 protection against beta amyloid toxicities presented its problems. In addition, MTT assay is not feasible in detecting cell viability in cell systems where gluthathione-S-transferase are upregulated as this enzyme directly affects MTT reduction. 311 Alamar Blue, or resazurin, 312 proved to be a sensitive measure for cell viability. 313 Alamar Blue use in kinetic assays is confounded by the phenolic moiety found in its structure and the high commercial, stock concentrations of 440 M. 312 In our hands, addition of Alamar blue concomitantly with oxidative stress-inducing insults offers protection against such insults (data not shown). Also, toxic insults such as sodium azide actually showed an increase in Alamar Blue reduction. 312 Calcein AM assay proved to be the best of the cell viability assays tested. Cell viability assessments using calcein AM correlated with the visual inspection of cells microscopically. Calcein AM has been used to determine cell viability in myocytes, 314 buccal mucosa tissue, 315 endothelial cells, 316 corneal tissue 317 and cells, 318 NG108-15 neuroblastoma-glioma cells, 319 hippocampal slice cultures, 320 and primary rat neurons. 290 Some resistance to its use assay as a viability marker has been made when copper and zinc were applied to the freshwater ciliate Tetrahymena pyriformis. 321 Quantification is performed using microplate readers or by manual counting for specific morphological criteria. In the latter case, propidium iodide can be added with calcein AM in living (that is, not artificially permeabilized by detergents) cells to differentiate between healthy and

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27 dying cells. Aside from cell viability assessments, calcein AM is also used to test mitochondrial permeability transition pertubations. 322 In this assay cells are loaded with calcein AM, the cytosolic and nuclear calcein fluorescence is quenched with cobalt resulting in only mitochondrial calcein fluorescence. Injury to cells lead to the opening of permeability transition pores and the release of mitochondrial calcein into the cytosol. Glutamate toxicity in HT-22 cells is a well-used paradigm 100,323-326 for oxidative stress-induced neuronal cell death and has been categorized as oxytosis by Tan et. al. 327 Glutamate toxicity in this cell model has been linked with glucocorticoid receptor nuclear localization 325,328 and has been exacerbated by glucocorticoids. This exacerbation is seen in other neuronal cell models. 329-332 That post-glutamate additions of estratrienes were protective is not all that compelling as glutamate does not kill cells until 10h; however changes in biochemical parameters do change (GSH decreases, ROS, etc) within hours. IAA is another insult used in cell culture 333-336 and animal models. Here we present for the first time E2 protection against IAA toxicity. IAA reduces ATP and phosphocreatine levels and mimics the acute effects seen in ischemic tissue damage. Also, in Huntington’s disease models, huntintin with polyglutamine repeats binds to GAPDH 337 and IAA produces energy impairment in the striatum. 301 Estratrienes respond to IAA in a similar manner to that of glutamate and that cysteine, N-acetylcysteine, and glutathione protected HT-22 cells from glutamate and IAA-toxicity alludes to an oxidative pathway. Indeed, others have found IAA-induced increases in reactive oxygen species 338 and specifically the hydroxyl radical. 301 These increases in ROS may explain the changes in membrane stability seen with IAA treatment. Wu et. al. 336 found that IAA addition to human astrocytoma cells results in rapid depletion of ATP and a concomitant

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28 decrease in membrane lipid order as determined by fluorescence anisotropy (r) and fluorescence recovery after photobleaching (FRAP) and loss of membrane integrity as assessed by propidium iodide uptake. E2 has been shown to bind specifically to GAPDH and was able to affect Vmax and Km for this enzyme. 339,340 In the presence and absence of IAA, E2 (10 M) did not affect GAPDH activity in our hands (data not shown). Steroid hormones have also been shown to interact with aldehyde dehydrogenase and glucose-6-phosphate dehydrogenases . 341 Since IAA is an alkylating sulfydryl-binding molecule, it may also affect other proteins with susceptible sulfydryl groups. Working with beta amyloid peptides proved cumbersome. There was interference with determining estrogen protection against these peptides, as estradiol exerted effects on the extrusion of the formazan dye, which had nothing to do with the inert toxicity of the peptide, itself. Also, in more cases than not, we did not find a dose-dependent relationship between the concentration of peptide and toxicity. At 24h, nM and M concentrations of beta amyloid peptides reduced MTT reduction by approximately 20%. We might detect dose-dependency if we incubated for longer periods of time. Also, the calcein AM assay was not able to consistently pick up A toxicity when used as a fluorescence microplate assay. It has been shown that manual counting using calcein AM was feasible. 290 It is of interest that we confirmed results from Shearman et.al. group 342 in that early time points with A peptides resulted in reduction in MTT activity, but no decrease in cell viability as determined by [3H]-thymidine uptake or release in lactate dehydrogenase into the cytosol in their experiments. It may be that the inability to detect A kill at 24h by calcein AM is an expected result. Others believe A inhibition of MTT reduction is not dependent on the mitochondria, but is dependent on A’s ability to

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29 increase the exocytosis of MTT from lysosomes/endosomes to the cell membrane. 343 On the other hand, Pereira et.al. 304 show A-induced decreases in complexII/III and complex IV mitochondrial activity. Estrogen concentrations needed for neuroprotection were in the pharmacological range. Indeed, hippocampal cells do not encounter the destructive magnitude that these insults incur. Since estrogen neuroprotection is dependent on the severity of the insult, it may be that the estrogen doses needed are less than that seen in these in vitro models. Also, estrogen levels measure the free, circulating and not the bound forms. The actual estrogen levels may be higher, although probably not to the same extent used here. These pharmacological levels, however, were attainable when conjugated equine estrogens (1.25 mg) were given as a single oral dose to postmenopausal women. 344 Likewise, the steroid compound RU486 reached micromolar concentrations after oral administration. 345 It may be that lower, chronic doses of estrogens could be protective.

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30 0 3000 6000 9000 12000 15000 18000 0.0 0.1 0.2 0.3 0.424 h 48 h MTT AssayCells/WellO.D. 0 3000 6000 9000 12000 15000 18000 0 1000 2000 3000 400024 h 48 h Calcein AM AssayCells/WellRFU 0 3000 6000 9000 12000 15000 18000 0 10000 20000 30000 40000 5000024 h 48 h Alamar Blue AssayCells/WellRFU Figure 3.1 Cell density versus assay response. HT-22 cells were seeded at the indicated concentrations and allowed to incubate for 24 or 48 hours. Three different viability assays, (A) MTT, (B) Calcein AM, and (C) Alamar Blue, were utilized to determine the appropriate concentration of cells to seed.

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31 051020 0 25 50 75 100 125MTT Calcein AM (3,000 c/w) Calcein AM (5,000 c/w) A(25-35) (M)% Control Figure 3.2 HT-22 cells were seeded at either 3,000 or 5,000 cells per well in 96-well plates. After overnight incubation, cells were treated with various concentrations of A(25-35) for 24 hours. Cell viability was determined by calcein AM assay and data are presented as percent of control.

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32 Calcein AM Assay 500100020004000800016000 0 1000 2000Control 20M IAA 10 mM Glut [Cells/well]RFU Calcein AM Assay 500100020004000800016000 0 25 50 75 10020M IAA 10 mM Glut [Cells/well]% Control Alamar Blue Assay 500100020004000800016000 0 2500 5000 7500 10000Control 20M IAA 10 mM Glut [Cells/well]RFU Alamar Blue Assay 500100020004000800016000 0 25 50 75 100 12520M IAA 10 mM Glut [Cells/well]% Control MTT Assay 500100020004000800016000 0.0 0.1 0.2 0.3Control 20M IAA 10 mM Glut [Cells/well]O.D. MTT Assay 500100020004000800016000 0 50 100 15020M IAA 10 mM Glut [Cells/well]% Controla.f.d.b.e.c. Figure 3.3 Effect of various insults on HT-22 cells seeded at different densities using three different cell viability assays. HT-22 cells were seeded at the indicated densities in 96-well plates and allowed to incubate overnight. Cells were treated with iodoacetic acid or glutamic acid for 16 h. Plates were assayed for cell viability using the Calcein AM assay (a,b), Alamar Blue assay (c.d), and MTT assay (e,f). Left-hand figures depict raw values (a,c,d) and right-hand figures depict normalized values (b.d,f) of n=4 wells per group.

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33 0 10 20 30 40 50 0 25 50 75 100Control 10M E2 [Glutamate]mM% Control 0 10 20 30 40 50 60 70 80 90 0 25 50 75 100Control 10M E2 [IAA]M% ControlAB Figure 3.4. Effect of E2 on glutamate and IAA-induced cell death. (A) HT-22 cells were cotreated with the indicated doses of glutamate and 10 M E2 for 16h and then assessed for cell viability using calcein AM. (B) HT-22 cells were cotreated with the indicated doses of IAA and 10 M E2 for 16h and then assessed for cell viability using calcein AM. Data are depicted as percent of control and represent mean SEM of n=4 wells per group.

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34 C A B D Figure 3.5 Photomicrograph of HT-22 cells. HT-22 cells were cotreated with glutamate and E2 for 16h. Calcein AM and propidium iodide were added for 15 min. Depicted are (A) Control, (B) 10 mM glutamate, (C) 10 M E2 and (D) glutamate and E2.

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35 1 2 3 4 5 6 7 8 9 0 25 50 75 100 125Control 10M IAA 20M IAA 40M IAA Hours% Control 5001000200040008000 0 25 50 75 10020 mM IAA Cells/well% Control Figure 3.6. Effect of IAA concentration and time on HT-22 cell viability. a) HT-22 cells seeded in 96-well plates (3500 cells per well) were treated with various doses of IAA for the indicated lengths of time and then assayed for cell viability using calcein AM. b) At 8h treatment, IAA cell death was dependent on cell seeding density. Values are expressed as percentage of controls and represent mean SEM of four wells per group.

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36 -4 -3 -2 -1 0 1 2 0 25 50 75 10024h Pre-tx Only Co-Treatment 5h Pre-tx Only 5 h Post-Treatment log [17E2]M% Control Figure 3.7. Pretreatment, Cotreatment, and Posttreatment effects of E2 on glutamate-induced cell death. HT-22 cells were E2 pretreated for 24 or 5h before glutamate, cotreated with glutamate, or given 5h after glutamate (10 mM) and assessed for cell viability 16h later using calcein AM. Data are depicted as percent of control and represent mean SEM of n=6 wells per group.

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37 -2 -1 0 1 2 3 4 0 25 50 75 100 12510 mM GLUT 20M IAA 40M IAA [Cysteine]M% Control -3 -2 -1 0 1 2 3 4 0 25 50 75 100 12510 mM Glutamate 20M IAA 40M IAA [DTE]M% Control Figure 3.8. Antioxidants and reducing agents protect against glutamate and IAA toxicity. (A) HT-22 cells were cotreated with cysteine and insults for 16h (glutamate) or 8h (IAA) and assessed for cell viability using calcein AM. (B) HT-22 cells were cotreated with the reducing agent, DTE, and insults as described in (A) and assessed for cell viability using calcein AM assay.

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38 02.5512.5 0 250 500 750 1000CONTROL 5 mM GLUT 10 mM GLUT [GLUCOSE] mMRFU*** 012.55 0 250 500 750 1000% FBSRFU**** Figure 3.9. Effect of glucose and serum concentration on glutamate-induced cell death. (A) HT-22 cells were cotreated with the indicated doses of glucose and glutamate for 16h and then assessed for cell viability using calcein AM. (B) HT-22 cells were cotreated with the indicated concentrations of serum and glutamate for 16h and then assessed for cell viability using calcein AM. Data are depicted as percent of control and represent mean SEM of n=4 wells per group.

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CHAPTER 4 STRUCTURE-ACTIVITY RELATIONSHIP OF ESTRATRIENES Introduction Estrogens are known neuroprotectants and have protected against many insults, such as hydrogen peroxide, A peptides, glutamate, heavy metals, and deprivation of growth factors. Many of these offenses involve an oxidative stress component and estrogens are known antioxidants. We 78 and others 99,346 have determined that the phenolic nature of the estradiol molecule is essential for neuroprotection. However, it would be beneficial to increase the neuroprotective potency of this therapeutic entity. We performed structure-activity relationship studies using rationally designed compounds in an established and a novel 17-estradiol-neuroprotection assay. We tested over 70 compounds in HT-22 (murine hippocampal) cells for their ability to inhibit cell toxicity against glutamate and iodoacetic acid, at two doses for each insult, and determined EC50 values to ascertain potency comparisons with E2 (chemical structure seen in Fig.4.1). In the present study, we test the hypothesis that estratrienes with a greater capacity to donate its phenolic electron (hydrogen) will better protect cells from oxidative stresses. We also determine whether hydrophobicity and planarity will affect the ability of estratrienes to protect cells from oxidative stress. Materials and Methods Cell culture HT-22 cells were maintained as described in Chapter 2. HT-22 cells were used in passes 15-30 and seeded at 3,000 to 5,000 cells per well in 96-well plates. After 39

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40 overnight incubation, estratrienes (concentrations ranging from 0.01 to 10 M) were coincubated with glutamate (10 and 20 mM) for ~16h or iodoacetic acid (20 and 40 M) for ~8h. Cell Viability Cell viability was assessed using the Calcein AM assay as described in Chapter 2. Data Analysis and Statistics All data are expressed as mean SEM. Each estratriene was tested in a mininum of at least three independent experiments (glutamate) or two independent experiments (IAA) with n=4-6 wells per experiment. In tables, data are expressed as EC 50 values (the concentration of estratriene that inhibits 50% of cell toxicity). The effects of estratriene inhibition of glutamate toxicity and IAA toxicity were determined by Spearman's rank order correlation analysis. The rank-order correlation coefficient, r s , is defined to be the linear correlation coefficient of the ranks. Results Estratriene Protection against Glutamate and IAA Toxicity As seen in Chapter 3, E2 (for structure see Fig. 4.1) was able to protect HT-22 cells against glutamate and IAA toxicity (Fig. 4.2a and 4.3a). Also shown are estratrienes (structures are pictured in Appendix A) that are potently protective (ZYC26, Fig 4.2c and 4.3c), ineffective (ZYC49, Fig. 4.2d and 4.3d), and similarly as potent (ZYC41, Fig. 4.2d and 4.3d) as E2. Correlations between Estratriene and Glutamate or IAA Toxicity As expected, the EC50 for high and low concentrations of each insult correlated with each other. In view of this finding that the SAR for 10 mM glutamate correlated with 20 mM glutamate (rs = 0.9218, 1/slope= 1.042, P value <0.0001), and that of 20

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41 M IAA with 40 M IAA(rs = 0.6008, 1/slope= 1.29, P value <0.0001) (Fig. 4.4), we present (discuss) data for the 10 mM glutamate and 20 M IAA sets. Estratriene protection against glutamate was comparable to that for protection against IAA. There was a positive correlation between 10 mM glutamate and IAA toxicity at 20 M (rs= 0.3567, 1/slope = 1.674, P value <0.0001) and 40 M (rs = 0.3930, 1/slope = 1.434, P values <0.0001) doses (Fig. 4.5 a,b). A similar result occurred with the 20 mM dose of glutamate and IAA toxicity (Fig. 4.5 c,d). Estradiol and Other Known Estratrienes 17-estradiol was able to protect HT-22 cells against 10 and 20 mM glutamate toxicities with an ED50 of 1.364 and 1.978 M, respectively. E2 was less effective in protecting HT-22 cells against IAA toxicity and required 2.902 and 4.704 M concentrations to inhibit 50% of IAA-induced cell death, at 20 and 40 M IAA, respectively (data not shown). Other known estrogenic compounds, estrone, DES, 17-estradiol, and ent-E2, were also able to inhibit oxidative stress-induced toxicity. The rank order for neuroprotection against 10 mM glutamate was entE2>E2>E1>E2>DES; and for 20 M IAA was DES>E1>E2>E2>entE2 (data not shown). A-Ring Derivatives A-ring derivatives with constituents that stabilized the phenoxy radical were better than E2 in protecting these cells from glutamate and IAA toxicity (Table 4.1). The synthetic strategy involved replacing hydrogen with a bulky group at the 2 or 4 positions. For those compounds that included the addition of a single group to the 2-carbon position of the A-ring (ZYC3, ZYC5, ZYC14, ZYC15, ZYC18, ZYC19, ZYC20, ZYC21,

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42 ZYC24, ZYC33, ZYC34, ZYC40, ZYC43, ZYC44, ZYC47) or 4-carbon position of the A-ring (ZYC16, ZYC17, ZYC48) the average EC50 value for protection against 10 mM glutamate and 20 M IAA was 0.817 +/0.478 and 0.491 +/0.136 M, respectively (Fig. 4.6). When two groups flanked the 3-OH position (ZYC22, ZYC 25, ZYC26), neuroprotection was enhanced. The EC50 values for protection against 10 mM glutamate and 20 M IA was 0.093 +/0.051 and 0.136 +/0.018 M, respectively (Fig. 4.6). The size of the bulky moiety could also influence the redox potential of the estratrience and minor groups (e.g. methyl) as well as highly symmetrical cage-like groups (adamantyl) were added to the 2 or 4 positions. There was no difference in neuroprotective potency of estratriene and the size of the bulky substituent (methyl, methyl-propyl, methyl-propenyl, tert-butyl, 3,3-dimethyl-butyl, 3,3-dimethyl-1-butynyl, and adamantyl) added. The methoxy ether analogue of ZYC5 (ZYC23) and ZYC 50 (ZYC 49) did not protect cells from death induced by glutamate and IAA. Further, ZYC23 potentiated the glutamate toxicity at the highest concentration tested (data not shown). Further negative controls included replacing the hydroxyl group at the 3 position of the A-ring with ethanolic or carboxylic groups (PS3 and PS4, respectively). These compounds were ineffective in protecting HT-22 cells from cell death. Extending the length of the steroid by adding a hydroxyl phenylethyl group to the 2-position of the A-ring (ZYC46) increased the neuroprotective properties. For example, ZYC46, with a long 4-hydroxyphenylethyl at the 2-position and thus contains two phenolic hydroxyl groups, performed slightly better against glutamate cell kill but only required approximately 4,000-fold less steroid to inhibit IAA induced toxicity. ZYC45, which has the 4-methoxy-phenylethyl addition at the C2 position, was more potent than

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43 E2 against both toxicities. Removing one of the two hydroxyl groups did not affect neuroprotection against glutamate, but decreased potency in the IAA model by approximately 300-fold. Displacing the phenolic hydroxyl group from the steroid backbone, thus extending the length of the molecule (ZYC28) was more protective than E2 against toxicity. Switching the hydroxyl group of the phenolic A-ring from the 3 position to the 2 position (ZYC37) decreased the potency against glutamate but not IAA toxicity. Switching the hydroxyl group to the 2 position as well as adding bulky groups to the 3 position (3-adamantyl, ZYC38; 3-tert-butyl, ZYC39), drastically improved neuroprotective potencies compared to that of ZYC37, an effect paralleling that seen comparing E2 with ZYC5 and ZYC15. B-and C-Ring Derivatives Hydroxyl additions to the B-ring (E2540, E2550) or C-ring (E1240, E2555, E2560) makes the normally lipophilic estrogens more hydrophilic. In all cases, whether above or below the plane of the ring, where hydroxyl groups were added to the B-and C-rings, neuroprotection was completely abolished. Opening of the B-ring (ZYC6 and ZYC7) results in decreasing the rigidity of the molecule. Opening the ring structure of estratrienes did not further prevent IAA-induced cell death as ZYC6 and ZYC7 performed equally compared to E2. On the other hand, ZYC6 and ZYC7 performed poorly as compared to E2 against glutamate, needing 6-12X higher concentrations to inhibit this toxicity by 50%. ZYC9 induces a nonplanar conformation in that the A and B rings lie perpendicular to the C and D rings. This change in the planarity of the molecule did not enhance or hinder neuroprotection.

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44 Reductions, removing hydrogens, from the ring structure is yet another way to increase the stability of the phenoxy radical. ZYC1, ZYC10, ZYC12, and ZYC27, for example, were more potent than E2 against oxidative stress toxicity. As a group, they were 179 and 488-fold more potent against glutamate and IAA toxicity, respectively. Reductions made to the B-ring, seen in conjugated equine estrogens, with intact phenolic hydroxyl groups (E380 and E400) were more potent against IAA toxicity, but performed equally as well against glutamate toxicity when compared to E2. Interestingly, the 3-O-methyl ether analogue of E400 (E430) showed modest neuroprotective effects against IAA toxicity, but as expected did not protect against glutamate toxicity. D-ring Derivatives The addition of benzoate (ZYC30), norcholesta (ZYC29), and norpregna (ZYC35 and ZYC36) to the 17-oxygen and pentanyl groups to the 16 carbon of the D-ring (ZYC2 and ZYC4) did not enhance the neuroprotection potency. Norcholesta, norpregna, and benzoate additions to the 17-position decreased neuroprotective potency against glutamate cell kill. Complete removal of a 17-substituent (ZYC13), however, enhanced neuroprotection. For PS1, the 17-hydroxy and 13-methyl groups results in a boat conformation which leaves the steroid relatively planar. For PS2, the 17-hydroxy and 13-methyl groups encourages a chair configuration which places the D-ring orthoganol and above the A,B,C-rings. These changes reduced potencies against glutamate and IAA-induced cell death. ZYC9, which also forms a boat/chair configuration was as effective as E2 in these neuroprotection schemes.

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45 Compounds which lack the steroid backbone, yet retain a hydroxy phenolic group (ZYC51ZYC59) were as neuroprotective as the molecules with the steroid nucleus. Discussion The present study expands the findings of Green 78 and Behl 99,346 in that estratriences are neuroprotective agents and demonstrates that compounds that have electron donating substituents, which increase the redox potential of the phenoxy radical, provide better neuroprotective abilities. The donated hydrogen radical can quench free radicals formed in oxidative stress conditions. Bulky substituents added to the A-ring and conjugated bonds introduced into the B and C rings accomplished this goal by increasing phenoxy radical stability by resonance stabilization. Polar groups added to the Band C-rings, disturbed estrogen’s ability to protect cells from oxidative stress. These hydroxyl groups, which impart hydrophilicity to the molecule, added to the middle of the steroid may impact the way these steroids fit into the center, hydrophobic lipid bilayer and thus its ability to react with lipid oxidation events. D-ring substituents also decrease the lipophilicity, but addition of norpregna, norcholesta, benzoate and methyl-ethers to the 17-position did not hinder neuroprotection, although these changes did decrease the potency. Pentanyl groups to the C-16 position decreased neuroprotection and may be due to the fact that the pentanyl group lies orthoganol to the A,B,C,D-rings and would thus impede the ability of the estratriene to situate in the center of the lipid membrane. Protection required a phenoxy hydroxyl group as the 3-O-methyl congener of ZYC5 and the O-methyl congener of ZYC50 proved ineffective. The hydroxy-phenol group did not have to be part of the A-ring as addition of a hydroxyl phenol group to the 3 position was just as, if not better, protective against both toxicities. The molecule is still

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46 planar and thus fits into the membrane. Repositioning the hydroxyl in the 2-carbon of the A-ring (ZYC37) was still, albeit less, protective against glutamate toxicity and was a better protector against IAA cell toxicity. This and the fact that the nonsteroidal compounds with phenolic groups were still protective shows that the position of the hydroxy phenol group is not restricted to the 3-position of the steroid backbone. Green et.al. 78 found that nanomolar concentrations were neuroprotective against serum deprivation in SK-N-SH neuroblastoma cells. In this case estrogens may work as neurotrophic agents. However, in concert with glutathione, estrogens were also potent neuroprotectants (in the nanomolar range) against A(25-35) toxicity in SK-N-SH 208 and HT-22 cells. 347 Using the same paradigm as Behl’s group, 99 we found that E2 was protective against glutamate toxicity at 10 M concentration. All studies found that the phenolic A-ring was an absolute requirement for neuroprotection, as compounds such as mestranol and quinestrol did not protect in their assay systems. That EC50 values seen with some of the more potent compounds were in the nanomolar range is key for therapeutic purposes. Although E2 is the only steroid with a phenolic A-ring, other steroids (progesterone, DHEA, testosterone) and non-steroidal hormones (melatonin, etc) have been shown to be neuroprotective in various injury paradigms. Green (PSG dissertation) did not find protection against serum deprivation in SK-N-SH cells up to 200 nM,. Behl did not find 10 M progestrone (P4) to be neuroprotective against glutamate insult in HT-22 cells. On the other hand, Gursoy 325 found optimal protection with 500 nM pregnanolone (a steroid with a hydroxyl group in the A-ring, but not a hydroxy phenol group) and no protection at 10M dose against glutamate insult in HT-22 cells. P4,

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47 however, has been found to be protective in acute global cerebral ischemia, 348,349 excitotoxicity in primary hippocampal cells, 86 and traumatic brain injury. 3,61,350 P4’s neuroprotective abilities in TBI may be due to its effects on edema. Progesterone derivatives have been shown, like E2, to elicit fast nongenomic events. There have also been several studies on the androgens, testosterone and DHEA, on neuroprotection. Some have determined a deleterious effect, while others a neuroprotective effect (DHEA: HT-22/ glutamate; 328 primary rat hippocampal cells/NMDA and kainate 351 ). Validation for neuroprotection by testosterone, however, necessitates the use of aromatase inhibitors as it is a precursor to estradiol. 352 Other estrogen-like substances, such as conjugated equine estrogens, 353 phytoestrogens, 246,354,355 and estrogen metabolites (2-OH-estradiol) 356 were also found to be neuroprotective, although not as potent as the compounds screened in these studies. Spearman’s rank correlations revealed significant relationships between protection against glutamate and IAA-induced cell toxicities. Correlations may be approved upon if the efficacy of the compounds were under consideration in the statistical analysis. Further correlations to explore would involve neuroprotection data against the direct chemical quantification of phenoxy radical stability. Nonetheless, we have ascertained the critical feature of the estradiol structure, stabilization of the phenoxy radical state, that enable increased potency against oxidative stress damage, thus advancing the knowledge base for estrogen neuroprotection.

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48 Figure 4.1 Structure of 1,3,5 (10)-estratriene-17-estradiol (E2). 17 13 A 2 3 4 5 16 15 12 11 14 8 7 6 9 B 10 C D 1 Figure 4.1 Structure of 1,3,5 (10)-estratriene-17-estradiol (E2).

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49 V.01.1110 0 250 500 750 1000CONTROL 10 mM GLUT 20 mM GLUT [17E2]MRFU V0.010.1110 0 400 800 1200CONTROL 5 mM GLUT 10 mM GLUT [ZYC-41]MRFU V0.010.1110 0 500 1000 1500 2000 2500CONTROL 10 mM GLUT 20 mM GLUT [ZYC26]MRFU V0.010.1110 0 100 200 300 400 500Control 10 mM GLUT 20 mM GLUT [ZYC49]MRFUabcd Figure 4.2. Examples of estratriene-mediated neuroprotection against glutamate-induced cell death. HT-22 cells were cotreated with estratriene and glutamate for 16 h and assessed for cell viability using the calcien AM assay. Shown are raw values of the mean SEM for n=4 per treatment group.

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50 V0.010.1110 0 250 500 750 1000Control 20M IAA 40M IAA [17E2]MRFU V0.010.1110 0 1000 2000Control 20M IAA 40M IAA [ZYC41]MRFU V0.010.1110 0 500 1000 1500Control 20M IAA 40M IAA [ZYC26]MRFU V0.010.1110 0 1000 2000Control 20M IAA 40M IAA [ZYC49]MRFUabcd Figure 4.3. Examples of estratriene-mediated neuroprotection against IAA-induced cell death. HT-22 cells were cotreated with estratriene and glutamate for 8 h and assessed for cell viability using the calcien AM assay. Shown are raw values of the mean SEM for n=4 per treatment group.

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51 0 10 20 30 40 50 60 70 0 25 50 75Rank Order (Glut; 10 mM)Rank Order(Glut; 20 mM) 0 10 20 30 40 50 60 70 0 25 50 75Rank Order(IAA; 20M)Rank Order(IAA; 40M) Figure 4.4 Spearman’s rank correlations between two doses of glutamate and two doses of IAA. 0 10 20 30 40 50 60 70 0 25 50 75Rank Order (Glut; 10 mM)Rank Order(IAA;0M) 0 10 20 30 40 50 60 70 0 25 50 75Rank Order (Glut; 10 mM)Rank Order(IAA;40M) 0 10 20 30 40 50 60 70 0 25 50 75Rank Order (Glut; 20 mM)Rank Order(IAA;0M) 0 10 20 30 40 50 60 70 0 25 50 75Rank Order (Glut; 20 mM)Rank Order(IAA; 40M)abcd Figure 4.5. Spearman’s rank correlations for glutamate and IAA neuroprotection data.

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52 GLUT(10)GLUT(20)IAA (20)IAA(40)C2C3C4ZYC30.1590.3660.216adamantyl-OHZYC50.1230.1210.1200.152adamantyl-OHZYC140.2150.2400.1830.382tert-butyl-OHZYC150.0470.8030.6461.200tert-butyl-OHZYC160.8820.8602.2501.912-OHmethyl-propenylZYC170.2440.2700.6800.828-OHmethyl-propylZYC180.5690.9210.2641.044methyl-propenyl-OHZYC190.1840.2720.0990.302methyl-propyl-OHZYC200.1320.2730.9081.134methyl-propenyl-OHZYC210.1220.2450.1080.282methyl-propyl-OHZYC220.0300.0300.0840.358adamantyl-OHmethyl-propylZYC23npnpnpnp-OCH3ZYC240.6650.7681.1323.928-OHmethylZYC250.2380.1470.1590.359t-butyl-OHmethylZYC260.0120.0370.1640.280adamantyl -OHmethylZYC280.4970.5680.4581.2683-p-hydroxy-phenyl-OHZYC330.0940.1270.0460.086adamantyl-OHZYC340.1210.1900.0460.219tert-butyl-OHZYC380.1330.2130.0210.035adamantyl2-OHZYC390.1800.3680.0490.282tert-butyl2-OHZYC400.5240.443adamantyl-OHZYC4111.3001.5731.341Iodine-OHZYC421.1671.5100.0510.942-OHIodineZYC430.7301.5111.3931.4493,3-dimethyl-1-butynyl-OHZYC440.3630.3990.0510.1673,3-dimethyl-butyl-OHZYC450.6840.9750.2390.4464-methoxy-phenylethyl-OHZYC460.8660.4650.0690.1554-hydroxyphenylethyl-OHZYC470.1390.1210.0170.088tert-butyl-OHZYC480.1790.5270.1761.599-OH3,3-dimethyl-butylZYC49npnpnpnp[3,2-b]furan, 4-methoxyphenylZYC500.1110.1390.0810.235[3,2-b]furan, 4-hydroxyphenylE430npnp7.92333.470-OCH3PS3npnpnpnp-CH2CH2OPS4npnpnpnp-COCH3 Table 4.1 Potencies of A-ring modified compounds.

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53 GLUT (10)GLUT (20)IAA (20)IAA (40) 0 5 10 15 202 or 4 No Addition 2 and 4 Average EC50 Values(M) Figure 4.6 Average EC50 values of A-ring substituted rings. EC50 values were averaged for those compounds that had one substitution added to either the 2 or 4 position, compounds that had two additions made to both the 2 and 4 positions, and compounds that did not fit in these two groups.

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CHAPTER 5 ANTIOXIDANT CAPACITY OF ESTRATRIENES Introduction Oxidative stress is an imbalance between the generation and detoxifcation of prooxidants in favor of the former. Reactive oxygen species (ROS) are endogenously produced during normal aerobic respiration (mitochondria), quinone metabolism, by peroxisomes (degradation of fatty acids and other molecules produce hydrogen peroxide as a by-product), and by the induction of cytochrome P450 enzyme which lead to oxidant by-products. In neuronal cells, H2O2 production comes from monoamine oxidase, tyrosine oxidase, and L-amino oxidase activities as well. Free radicals can originate from exogenous (environmental) sources as well: tobacco smoke, ionizing radiation, organic solvents, anesthetics, hyperoxic environments, and pesticides. Antioxidant defenses include enzymatic (superoxide dismutase, catalase, gluthathione peroxidase/reductase, etc.) and nonenzymatic (ascorbic acid, glutathione, uric acid, tocopherol, ubiquinol) soluble molecules. Excess oxidative stress leads to macromolecular damage to lipids, nucleic acids, and proteins and thus impairs normal cell function. The brain is more susceptible to oxidative stress. Twenty percent of cardiac output goes to brain and the brain is only 2 % of total body weight; i.e. the brain has a high oxygen consumption rate. There is a relatively higher amount of metals, a rich source of lipids (especially the polyunsaturated fatty acids) and a weaker antioxidant defense system. With an inability to regenerate neurons after insult, oxidative damage to brain is more daunting. It is, therefore, not surprising that oxidative stress has been implicated in 54

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55 a variety of neurodegenerative diseases 294 , including Alzheimer’s disease, 357 Parkinson’s disease, 298 and amyotrophic lateral sclerosis (mutations in superoxide dismutase (SOD-1). 358 Further, a prominent theory on aging implicates free radical reactions. 359,360 Reactive oxygen species are chemically reactive oxygen-containing molecules. These species can influence normal cellular processes by affecting intracellular signaling pathways. In extreme cases of oxidative stress, they can become deleterious to cell health. Lipid peroxidation is especially dangerous as removal of a hydrogen ion (oxidation) from a polyunsaturated fatty acid (PUFA) result in a hydroperoxy radical that leads to a chain reaction of events including the generation of organic peroxides, conjugated dienes, malondialdehydes, and 4-hydroxynonenal. Peroxidation of unsaturated fatty acids compromises the plasma membrane by making lipids more hydrophilic, thus disrupting membrane protein function of transporters, ion channels, and/or receptors. We have shown that estratrienes designed to increase donation of a proton from the phenolic rings protect neuronal cells lines from oxidative stress, thus serving as an antioxidant. 17-estradiol is an established antioxidant agent in many systems, including LDL oxidation, cholesterol oxidation, and conjugated diene formation. 361-363 For these reasons, we want to determine if modifications of the 17-estradiol structure would increase the antioxidant capacity. In this set of studies we determined whether these compounds could affect biochemical parameters related to oxidative stress, that is, to scavenge reactive oxygen species and to inhibit lipid peroxidation. Materials and Methods

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56 Cell Culture HT-22 cells were maintained as described in Chapter 2. HT-22 cells were used in passes 15-30 and seeded at 3,500 cells per well in 96-well plates. DCF Assay Relative levels of cellular ROS were measured using a fluorescent dye 2,7-dichlorofluorescin-diacetate (DCF) microplate assay. HT-22 cells were loaded with 50 M DCF dye for 45 min, washed twice with PBS, and subsequently treated with insults (50 M hydrogen peroxide or 2 U/ml glucose oxidase (GO)) with or without a 1 h pretreatment with estratrienes. TBARS Lipid peroxidation was determined using the TBARs assay. Malondialdehyde is an end product of lipid peroxidation; it is derived from the breakdown of polyunsaturated fatty acids (PUFAs) and related esters. Rat brain homogenates were subjected to iron chloride (50 M) for 15 minutes. Estratrienes were added to assess for their ability to inhibit this stimulated peroxidation event. IC50 values represent the concentration of estratriene needed to reduce LPO by 50%. Statistics Results are expressed as mean SEM, and statistical significance was determined by ANOVA followed by post-hoc Tukey’s test. A p value of less than 0.05 was considered significant. Spearman’s rank correlation was used to determine a relationship between inhibition of lipid peroxidation and neuroprotection. Results

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57 Effect of Glucose Oxidase on Production of Reactive Oxygen Species The flavoenzyme, glucose oxidase (GO), catalyzes the following reaction: -D-glucose + O 2 + H 2 O D-gluconolactone + H 2 O 2 . In HT-22 cells, GO dose-dependently increased reactive oxygen species up to 625 mU/ml and had an EC50 of 308.3 mU/ml (Fig. 5.1a). Hydrogen peroxide dose-dependently increased ROS levels between 12 and 750 M concentrations and had an EC 50 of 100.6 M (Fig. 5.1b). Effect of Estratrienes on Scavenging Reactive Oxygen Species In HT-22 cells, E2 did not significantly affect glucose-oxidase mediated increases in ROS (Fig. 5.2a). In SK-N-SH cells, higher concentrations of estratrienes inhibited H2O2-induced increases in ROS (Fig. 5.2b). The more potent neuroprotectant, ZYC26, was a better scavenger of ROS than E2. However, ZYC26 scavenger ability was still quite weak in this regard. Effect of Iron Chlorideinduced Lipid Peroxidation on Homogenates Standard curves using 1,1,3,3-tetramethoxypropane standards ranging from 0 – 10 M were prepared for assessment fluorometrically and spectrophotometrically. Standard curves read fluorometrically with excitation wavelength of 530 nm (bandwidth 25), emission wavelength of 590 nm (bandwidth 20) and at a sensitivity of 100 produced a linear curve with an r2 = 0.9981 (Fig. 5.3a). When the same plate was read at an absorbance wavelength of 560 nm the r2 = 0.8276 (Fig. 5.3b). Iron dose-and time-dependently induced lipid peroxidation in rat brain homogenates (RBH) when incubated with 5-500 M Fe3+ for 15’, 30’, 1h and 2h (Fig. 5.4a). HT-22 and C6 glioma homogenates required higher concentrations of iron for dose-and time dependence of lipid peroxidation (Fig. 5.4b,c).

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58 Effect of Estratrienes on Iron-Induced Lipid Peroxidation Estratrienes were able to inhibit iron-induced lipid peroxide production. In relation to the other estratrienes, estradiol and estrone similarly inhibited lipid peroxidation, but required higher micromolar concentrations to inhibit oxidation of lipids (Fig. 5.5). ZYC49, an estratriene that lacks a hydroxylated phenol ring and did not protect in neuroprotection assays, did not inhibit lipid peroxidation (Fig. 5.5). ZYC26, which has two groups that flank the 2-hydroxyl group of the A-ring, potently inhibited lipid peroxidation and required only 700 nM to inhibit 50% of iron-induced lipid peroxidation (Fig. 5.5). ZYC41, which has iodine at the 2-position, performed better than E2 with an EC50 of 6.291 M in this model system. Correlation Between Inhibition of TBARs and Glutamate and IAA Protection The potency of the estratrienes for protection against glutamate and IAA-toxicity were compared with the antioxidant capacity of these compounds to inhibit peroxidation events. There was a positive correlation between inhibition of iron-induced lipid peroxidation and neuroprotective potency in the glutamate and IAA-induced cell death models. Inhibition of TBAR levels correlated with potencies for neuroprotection against 10 mM glutamate (Fig. 5.6a), 20 mM glutamate (Fig. 5.6b), 20 M IAA (Fig. 5.6c), and 40 M IAA (Fig. 5.6d), with r s values (1/slope, P values) of 0.6637 (1.227, <0.0001), 0.6483 (1.242, <0.0001), 0.3315 (1.737, <0.0001), and 0.5063 (1.405, <0.0001), respectively. When graphically depicted into categories of low, medium, and high EC50 values for TBARs inhibition the trend seen in lipid peroxidaton inhibition is paralleled in the neuroprotection assays (Fig. 5.7).

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59 Discussion The present study demonstrates that estratrienes are potent lipid antioxidants. This is a well-known property as previous studies have determined the radical scavenging, iron chelating, and total AO properties of various estratrienes. 364-366 We have confirmed other reports that show 2and 4-additions to the phenolic A-ring are potent antioxidants. 361 Further, they are able to do so in a pattern similar to their ability to protect HT-22 cells from oxidative stress-induced models of toxicity. In the DCF assay, levels of ROS were detected by the direct bolus addition of hydrogen peroxide and an enzyme-mediated method. Glucose oxidase and hydrogen peroxide-mediated increases in ROS reached a plateau. Additional glucose (substrate for GO) was not coadministered with the enzyme, and this might account for the plateau seen. There could also be a self-quenching effect. E2 was not able to significantly decrease ROS when HT-22 cells were insulted with glucose oxidase. When higher concentrations of estratrienes were used, we were able to see a scavenging effect in SK-N-SH cells. In a cell free system, we have shown that estratrienes are able to inhibit iron-induced lipid peroxidation. Although the scavenging abilities for soluble ROS are not robust as seen in the DCF assay. Estratrienes performed better in the lipid-based antioxidant model, TBARs assay. This occurs in spite of the study showing that antioxidant capacities of various estrogenic structures were found to be dependent upon the aqueous or lipophilic nature of the assay system. 367 In the aqueous-based antioxidant assay, phenolic estrogens performed better than catecholestrogens and diethylstilbestrol in quenching the chromogenic radical cation 2,2’-azinobis-(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS+). On the other hand, in the lipid-based antioxidant system

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60 catecholestrogens were able to regenerate -tocopherol for oxidative stress-treated LDL while phenolic estrogens could not. Nevertheless, we confirmed that E2 is able to inhibit lipid peroxidation in rat brain homogenates with an IC50 of 19.83 M, which is comparable to the IC50 seen by other groups (IC = 21 M). 206 Ruiz-Larrea et.al. 207 found that catecholestrogens and DES increased the rate of Fe(III) reduction in aqueous solutions, while E2 and E1 did not affect this rate. If LPO is based on Fe (III), this corroborates with our findings in that DES was better able to decrease MDA formation compared to E2 and E1: DES (6.7)
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61 Oxidative stress is of major pathophysiological relevance for a variety of process (cancer, ischemia-reperfusion, atherosclerosis, and cataractogenesis) and as well as for neurodegenerative diseases (AD, PD, ALS). Since an imbalance of the production and elimination of ROS, with production winning out cause OS, it would be imperative to counteract this by increasing the elimination process or preventing ROS production. There are two ways to combat this: enzymatically and non-enzymatically. The use of antioxidants that penetrate the blood-brain barrier could alleviate oxidative damage in the brain. Estrogens are known antioxidants and with its hormone structure it can easily get into brain.

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62 0204080160300625125025005000 0 500 1000 1500 2000[GO] mU/mlRFU 011.7223.4446.8893.75187.50375.00750.001500.003000.00 0 2500 5000 7500 10000[H2O2]MRFU Figure 5.1. Effect of glucose oxidase and hydrogen peroxide on reactive oxygen species levels as detemined by DCF Assay. HT-22 cells loaded with DCF-DA were treated with (A) glucose oxidase or (B) hydrogen peroxide and assessed for ROS 1h later. Depicted are mean SEM for n=4-7 wells.

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63 V0.111020 0 200 40017E2 (M)% Increase ROS Levels Figure 5.2 Effect of estratrienes on ROS. (A) E2-treated HT-22 cells were incubated with glucose-oxidase and assessed for ROS 1h later. (B) E2 or ZYC26 (6-50 M) were incubated at the indicated concentrations in SK-N-SH cells for 1h, treated with hydrogen peroxide and then assessed for ROS scavenging 1h later. Values are expressed as percent increase in ROS levels and represent the mean SEM of 5-7 wells per group.

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64 0 2 4 6 8 10 12 0 2000 4000 6000[TEP]MRFU 0 2 4 6 8 10 12 -0.05 0.00 0.05 0.10 0.15[TEP]MO.D. (560 nm)ab Figure 5.3. MDA standard curves assessed (a)fluorometrically and (b) spectro-photometrically. Rat Brain Homogenates 0550500 0 2500 5000 7500 1000015' 30' 1h 2h [FeCl3]MRFU HT-22 Homogenates 0550500 0 250 500 750 100015' 30' 1h 2h [FeCl3]MRFU C6 Glioma Homogenates 0550500 0 100 200 30015' 30' 1h 2h [FeCl3]MRFUABC Figure 5.4. Effect of iron chloride on homogenates of (A) rat brain, (B) HT-22 cells, and (C) C6 glioma cells. Cell or tissue homogenates were treated with the indicated concentrations of FeCl3 for 15’, 30’, 1h, or 2h and then assessed for MDA levels fluorometrically. Depicted are mean SEM for n=6 wells.

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65 -3 -2 -1 0 1 2 0 1000 2000 3000 4000 5000E2 ZYC26 ZYC49 E1 ZYC41 log CONCENTRATION (M)RFU Figure 5.5 Estratriene-mediated inhibition of iron-induced lipid peroxidation. Rat brain homogenates were preincubated with the indicated steroids for 30 minutes. Iron was then added for another 15 minutes. Levels of TBARs were measured fluorometrically. Depicted are mean SEM for n=3 wells.

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66 a b c d Figure 5.6 Correlation between inhibition of iron-induced increases in rat brain homogenates and neuroprotection potency in glutamate and IAA neuroprotection models. Neuroprotection data were obtained from Chapter 3.

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67 GLUT (10)GLUT (20)IAA (20)IAA (40)TBARs 0 10 20 30< 2M 2-10M > 10M Average EC50 Values(M) Figure 5.7. Graphical representation the relationship between lipid peroxidation and neuroprotection. Data were categorized into three groups: those that had EC50 values for TBAR inhibition below 2 M, those compounds with EC50s between 2 and 10 M, and those compounds requiring greater than 10M concentrations to inhibit lipid peroxidation by 50%.

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CHAPTER 6 ESTROGEN RECEPTOR INVOLVEMENT Introduction Estrogens are known neuroprotective agents, but are not without health risks. 369,370 To be considered as therapeutic agents, enhancement of neuroprotective activity is not enough as it is also imperative that these molecules not elicit carcinogenic effects. To eliminate estrogenic effects, i.e. carcinogenicity, one can introduce chemical modifications to the estradiol pharmacophore that inhibits its affinity for the estrogen receptor. The estrogen receptors are ligand-activated transcription factors that belong to the steroid hormone receptor family. In the classical scheme of estrogen mechanism of action, 371,372 estrogens travel through the bloodstream attached to sex hormone binding proteins. They diffuse through the plasma membrane and bind to estrogen receptors. This binding causes a conformational change, which results in the dissociation of heat shock proteins. A pair of E2/ER form dimers and then binds to the estrogen response element (ERE). Interaction with basal transcription factors leads to transcription of ERE-regulated genes. Tamoxifen is a mixed agonist/antagonist of estrogen receptors (ER) that is used clinically for secondary prevention of breast cancer. 373,374 ICI 182,780 (7-[9-(4,4,5,5,5,5-pentafluoropentyl-sulfinyl)nonyl)estra-1,3,5(10)-triene-3,17-diol) is believed to be a pure ER antagonist. 375 Both compounds block the ER with IC 50 s in the nM range 375,376 and at similar concentrations inhibit growth of mammary tumor cells. 375 68

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69 As such, these compounds have been used in a variety of in vivo and in vitro studies to determine ER involvement in estrogen effects. In particular ICI 182,780 has become the “gold standard” for assessment of ER-mediated effects of estrogens. 377-385 The present study was undertaken to determine the estrogenic activity of neuroprotective estratrienes by testing whether estrogen receptor antagonists can block neuroprotective effects and whether these estratrienes can bind the estrogen receptors. Materials and Methods Cell Culture HT-22 cells were maintained as described in Chapter 2. HT-22 cells (passages 18-25) were seeded into Costar 96-well plates (Corning, NY) at a density of 5,000 cells/well and treated the next day. SK-N-SH cells were maintained in the same manner as HT-22 cells with the exception of the media used, RPMI-1640 (Gibco-BRL). Cells used in these experiments were from passages 38-47. Cells were plated at a density of 12,000 cells/well and treated 48 h after seeding. Experiments were initiated by the addition of ICI 182,780 (Tocris, Ballwin, MO), tamoxifen (Sigma) or 4-hydroxytamoxifen (Tocris) in concentrations ranging from 0.1 to 100 M. For all cell cultures, compounds were added to the media without a media change. Exposure times ranged from 24 to 72 h. Cell Viability Cells were insulted for ~16 hours after which cell viability was determined by calcein AM assay as described in Chapter 2. Estrogen Receptor Binding Assay Competition binding assays were performed using a commercially available kit, HitHunter EFC Estrogen Chemiluminescence Assay kit (Fremont, CA). This assay

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70 uses an enzyme fragment complementation (EFC) method. In brief, competing ligands at final concentrations ranging from 10 pM to 10 M were incubated with 5 nM recombinant estrogen receptor (ER) or 10 nM estrogen receptor (ER) (Panvera, Madison, WI) and 17-estradiol-conjugated enzyme donor (ED) for 1.5h. The enzyme acceptor (EA) was then added for another 1.5 h after which chemiluminescence substrate was added for another 1h. Relative luminescence units (RLU) were determined using a Biotek FL600 plate reader (sensitivity set at 150). Sigmoidal standard curves were created using a 4-paramater log curve with constant top and used to determine IC50 values (GraphPad Prism, version 3.02 for Windows, GraphPad Software, San Diego, CA). IC50 values for 17-estradiol in this assay system was 3 nM and 5 nM for ER and ER binding, respectively and these values for ER binding were found to be equivalent to those reported by the manufacturer. Data Reduction For viability estimates using the calcein AM assay, raw data were obtained as relative fluorescence units (RFU). All data were then normalized to % cell death, as calculated by (control value insult value)/control value x 100. Statistical Analysis All data are presented as mean SEM. The significance of differences among groups was determined by one-way ANOVA with a Tukey’s multiple-comparisons test for planned comparisons between groups when significance was detected Spearman’s rank correlation was used to determine a relationship between estrogen receptor or binding and neuroprotection or inhibition of iron-induced lipid peroxidation. For all tests, p < 0.05 was considered significant.

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71 Results ICI 182,780 Toxicity in HT-22 and SK-N-SH cells In HT-22 cells, ICI 182,780 toxicity was dose-dependent and toxicity appeared to occur during the first 24 h of exposure to the ER antagonist (Fig. 6.1a). Significant neurotoxicity was observed at concentrations as low as 0.01 M and a dose-dependent increase in toxicity was seen through 10 M. Additional exposure time did not substantially increase this toxicity (data not shown). In SK-N-SH cells, slight, but significant toxicity was seen with ICI 182,780 at concentrations ranging from 0.01 to 10 M (Fig.6.1a). Tamoxifen Toxicity in HT-22 and SK-N-SH cells Tamoxifen had no toxic effect on HT-22 cells through 1 M, then killed 78% of cells by 24h at 10 M (Fig.6.1b) and essentially 100% of cells at 50 M (data not shown). Treatment for 48 h did not change the profile of the neurotoxicity dose-response curve (data not shown). Similarly, SK-N-SH cells were not adversely affected by tamoxifen through 1 M, but toxicity was seen at 10 M (Fig. 6.1b). 4-Hydroxytamoxifen Toxicity in HT-22 and SK-N-SH Cells For both HT-22 and SK-N-SH cells, 4-hydroxytamoxifen exerted no toxicity through 1 M, but killed 68 and 51% of HT-22 and SK-N-SH cells, respectively, at 10 M (Fig.6.1c.). Antagonists Effect on Cell Insults ICI 182,780 was protective against 10 mM glutamate in HT-22 cells and reduced toxicity at 1 and 10 M by reducing the percentage of cell death from 69.0 1.7 to 57.5

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72 3.1 and 37.6 2.1, respectively (Fig. 6.2a). By contrast, tamoxifen potentiated 20 mM glutamate kill at 0.1 and 1 M by increasing the percentage of cell death from 45.3 2.3 to 74.8 1.8 and 78.1 3.5, respectively (Fig. 6.2b). In SK-N-SH cells (which are insensitive to glutamate toxicity), ICI 182,780 modestly improved cell loss due to serum deprivation from 74.7 4.3 to 63.5 4.0 cell death at 10 M (Fig. 6.2c). Tamoxifen had no significant effect on cell loss from serum deprivation ranging from 0.01 to 1 M but increased serum deprivation-induced cell death to nearly 100% at the 10 M concentration (Fig. 6.2d). Estrogen Receptor Binding Competition binding experiments revealed that E2 bound to ER and ER with EC50 values of 3.04 and 4.51 nM, respectively (Table 6.1). DES, as expected, bound slightly better and had EC50 values of 2.62 nM for ER and 1.84 nM for ER. Estrone, 17E2, and ent-estradiol bound to these receptors with less affinity (Table 6.1). Representative dose-response curves for known estrogens (estrone and 17-estradiol) and A-ring, adamantyl-possessing estratrienes (ZYC5 and ZYC25) are depicted for ER (Figure 6.3a) and ER (Fig. 6.3b) binding. Reductions made to the steroid structure (ZYC1, ZYC 10, ZYC27, E380) did not drastically change the affinity of the estratriene for the estrogen receptors, exceptions being with ZYC12 and E400 (see Appendix B for tables). Additions to the A-ring drastically affected binding to the estrogen receptors. Additions to the 2and 4-positions (ZYC 22, ZYC25, ZYC26) completely abolished the affinity of estratriene for both estrogen receptors. Adamantyl groups added to the 2 position (ZYC3, ZYC5, and ZYC26), likewise, abolished binding to the estrogen receptors. Methylpropenyl,

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73 methylpropyl, tert-butyl additions to the 2-position of the A ring greatly reduced the binding affinity for the receptors. Midsize additions (tert-butyl) to the 2-position and norpregna additions to the other end of the estratriene (ZYC47) further reduced the binding capacity. While bulkier additions (methyl propenyl (ZYC16) and methyl propyl (ZYC17)) to the 4-position also completely inhibited estrogen receptor binding, smaller additions (methyl, ZYC24) were approximately 69 and 58-fold less effective in binding to the estrogen receptors. Hydroxyl additions to the B-and C-rings in the alpha or beta positions inhibited binding activity. Hydroxyl groups in the configuration to the C11 position and of the estradiol derivative (E2560) bound 10-fold less to ER than 17-estradiol; the estrone derivative (E1240) bound with even less affinity. Changes to the planarity of the steroid ring structure also brought changes to the affinity for estrogen receptors. Opening the ring structure at the 9-position (ZYC6 and ZYC7) diminished the affinity of the steroid for both estrogen receptors; the estrone derivative (ZYC6) bound with less affinity than the estradiol derivative (ZYC7). The nonsteroidal compounds (ZYC51-59) bound with neglibible affinity. Correlation between Inhibition of TBARs and Glutamate and IAA Protection The potency of the estratrienes for ER binding was compared to protection against glutamate and IAA-toxicity as well as antioxidant capacity of these compounds to inhibit peroxidation events (see Appendix C for Spearman’s rank correlation values). In all scenarios, ER or ER binding presented a slight negative correlation with neuroprotective protection (Fig. 6.4 and Fig. 6.5) and lipid peroxidation inhibition (Fig. 6.6). When graphically depicted into categories of low, medium, and high EC50 values

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74 for ER binding, the trend seen in lipid peroxidaton inhibition and neuroprotection is in stark constrast to ER binding.(Fig. 6.7). Discussion The present report documents that both ICI 182,780 and tamoxifen are neurotoxic to a variety of neuronal cell types. In view of these data, the wide use of ICI 182,780, at the doses shown herein to be neurotoxic, as a “gold standard” to assess for the ER-dependence of neuronal estrogen responses appears unwarranted. This is particularly the case when neuroprotection is the measured estrogen response. In studies aimed at defining ER involvement in E2-induced neuroprotection, there are reports that ICI 182,780 antagonizes E2 effects, 378,380-383,386 that ICI 182,780 does not antagonize E2 effects, 377,385,387-389 and reports of a protective effect of ICI 182,708 alone. 209,377,390 With the exception of the reports by Brinton et al. 388 and Gridley et al. 377 who used 1 nM and 200 nM ICI 182,780, respectively, all other studies used ICI 182,780 at concentrations of 1 to 10 M. These concentrations were neurotoxic in HT-22 cells and SK-N-SK cells, were protective against glutamate toxicity in HT-22 cells (data not shown), were greater than the ED 50 for binding to the progesterone receptor, 391,392 and inhibit critical membrane associated proteins. 393,394 395 Given these reports of the lack of specificity of these doses of ICI 182,780 and the inherent toxicity of ER antagonists and their capacity to enhance (act as a second insult) or reduce the toxicity of other agents, we can draw no conclusion about the ER mediation of E2 neuroprotection based on ICI 182,780 studies. Although tamoxifen does not have a phenolic group, it can be metabolized to 4-hydroxy-tamoxifen, which does contain a phenolic hydroxyl group and therefore possesses the pertinent antioxidant moiety.

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75 More than 95% of binding affinity for the ER is dependent on the aromaticity of the A-ring. Removing the phenolic hydroxyl group eliminates most of the binding affinity for estrogen receptors and thus decreases carcinogenicity, but it would be counterproductive, as this moiety is an absolute requirement for antioxidant capabilities and cell protection. A way to circumvent this dilemma is to mask the 3–OH group with bulky groups in the 2 and 4 positions. This is important for three reasons: (1) it creates electron delocalizaton, (2) it imparts hydrophobicity to the A-ring and thus orients it toward the center of the lipid bilayer where lipid peroxidation events occur, and (3) it abolishes estratriene binding to estrogen receptors. 396 Fortunately, we found the last of these three to be true in our competition binding experiment. The most potent neuroprotective estratrienes, compounds with 3-hydroxyl groups flanked by two groups (such as adamantyl moieties), did not bind to estrogen receptors. We also show a lack of correlation between neuroprotection and ER and ER binding. If anything, there is a slight negative correlation between ER binding and neuroprotection. Also, binding does not guarantee transcriptional activity as binders could have antagonistic effects. ERE reporter gene assays would be useful to answer this issue. Further, other reporter gene assays for the ARE, AP1, etc could elicit insights into other possible mechanisms of estratriene neuroprotection. Ongoing debate centers on ER mediation of estrogen neuroprotection. Some claim ER mediates estradiol neuroprotection in MCAO, 51 while others do not. 397 Other possible mediators for estrogen neuroprotection in cerebral ischemia include cerebral blood flow/vascular mechanisms, 398 blockade of APP-induced expression, 399 antioxidant mechanisms, or the ability to promote neurogenesis. That these estratrienes protected

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76 against oxidative stress in HT-22 cells, which lack functional nuclear estrogen receptors 177,326,347 and the lack in correlation between neuroprotection and binding suggests that estratriene-mediated neuroprotection against oxidative stress is not mediated via estrogen receptors. In conclusion, we found that large bulky groups to the A-ring, that were neuroprotective antioxidants, were devoid of estrogen receptor binding.

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77 0.01 0.1 1 100 -25 0 25 50 75 100HT-22 SK-N-SH ******ICI 182,780 (M)% CELL DEATH 0.1 1 100 -25 0 25 50 75 100HT-22 SK-N-SH **TAMOXIFEN (M)% CELL DEATH 0.1 1 100 -25 0 25 50 75 100HT-22 SK-N-SH 4-OH-TAM (M)% CELL DEATH**ABC Figure 6.1 Effect of estrogen receptor antagonists on HT-22 and SK-N-SH cell viability. Dose-dependent effects of (A) ICI 182,780, (B) tamoxifen, and (C) 4-OH-tamoxifen on HT-22 and SK-N-SH cell viability. Cells were exposed to the indicated concentrations of antagonists for 24 h and viability was determined using a calcein AM assay. * indicates P< 0.05 versus the 0 dose group. N = 15 to 37 per group. Depicted are means SEM.

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78 00.1110 0 25 50 75 10010 mM GLUTAMATE 20 mM GLUTAMATE ***[ICI 182,780]M% CELL DEATH 00.1110 0 25 50 75 10010 mM GLUTAMATE 20 mM GLUTAMATE **[TAMOXIFEN]M% CELL DEATH 00.010.1110 0 25 50 75 100[ICI 182,780]M% CELL DEATH 00.010.1110 0 25 50 75 100*[TAMOXIFEN]M% CELL DEATHABCD Figure 6.2 Effect of ICI182,780 and tamoxifen on cell toxicity. (A,B) HT-22 cells were assessed for cell viability after cotreatment with antagonist and glutamate. (C,D) SK-N-SH cells were deprived of serum and treated with antagonists. Depicted are means SEM with n=6-8 wells per group. * indicates P< 0.05 versus the 0 dose group.

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79 Compound ER (nM) ER (nM) 17E2 3.04 4.51 17E2 16.46 83.75 Ent-E2 24.65 5.64 Estrone 13.35 30.92 DES 2.62 1.84 Table 6.1 Table for EC50 values for ER and ER competition binding experiments with known estrogenic compounds.

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80 -12 -11 -10 -9 -8 -7 -6 -5 -4 0 300 600 900 1200E1 17E2 ZYC 5 ZYC25 log ConcentrationRLU -12 -11 -10 -9 -8 -7 -6 -5 -4 0 250 500 750 1000E1 17E2 ZYC 5 ZYC25 log ConcentrationRLUAB Figure 6.3 Effect of estratrienes on estrogen receptor binding. Estratrienes at concentrations ranging from 10 pM to 10 M were assessed for their ability to bind to ER (A) and ER (B). Depicted are means SEM with n=3 wells per group.

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81 Figure 6.4. Correlation between estrogen receptor binding and neuroprotection. potencies in glutamate and IAA-induced cell death models.

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82 Figure 6.5. Correlation between estrogen receptor binding and neuroprotection potency in glutamate and IAA cell death models.

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83 Figure 6.6. Correlation between ER and ER binding and inhibition of iron-induced lipid peroxidation.

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84 GLUT (10)GLUT (20)IAA (20)IAA (40)ERERTBARs 0 5 10 15 20 25< 100 nM 100-1000 nM >1000 nM Average EC50 Values(M) Figure 6.7. Graphical representation the relationship between ER binding, lipid peroxidation and neuroprotection data. Data were categorized into three groups: those that had EC50 values for ER binding below 100 nM, those compounds with EC50s between 100-1000 nM, and those compounds requiring greater than 1M concentrations to inhibit ER binding by 50%. The y-axis is the average EC50 value for those compounds that fit the above criteria.

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CHAPTER 7 SUMMARY AND FUTURE DIRECTIONS Males and females are different, more specifically when it pertains to brain structure/function, incidence and type of stroke, neurological disorders, autoimmunity, and the like are concerned. When females enter the menopause, however, their chances of neurological injury puts them at an equal pace with males for such conditions. The differences seen between males and females and that of premenopausal females and postmenopausal females are the presence or absence of ovarian hormones. Males do produce estrogens but their susceptibility to injury may involve other hormones. Further, with the aging of the population, more women will spend more time living in an estrogen-deprived state. Much research has purported the beneficial effects of estradiol on bone and cardiovascular health, but estrogens also affect both normal and pathological brain functions. The precipitous decline in estrogen levels with the menopause leaves the brain vulnerable to insults. These women must decide whether to take steroid replacement therapy. Unfortunately, recent Women’s Health Initiative (WHI) trials have found estrogen plus progestin (medroxyprogresteone acetate, to be exact) therapies to be more deleterious than protective in regards to stroke 400 and cognitive function. 401 With this recent scare imposed by the WHI studies and the resulting termination in the combined progestin and estrogen arm regimen, the need for estrogenic alternatives is warranted. The conjugated equine estrogens (CEE) used in the WHI trials (E380 and E400) were protective in our oxidative stress paradigms, albeit less potent than the A-ring derived 85

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86 estratrienes. These CEEs, however, were still able to bind to the estrogen receptors with high affinity. Estrogen analogues that retain the neuroprotective properties but lack the unwanted side effects (breast and uterine cancers, thromboembolysis, etc.,) would prove beneficial. Estrogen’s reputation as an antioxidant makes these molecules a therapeutic choice against the ravaging effects of oxidative stress seen in many neurodegenerative diseases, such as AD, PD, and ALS. We have shown estratriene protection seen in a glutamate-induced oxidative stress paradigm and corroborated their neuroprotective effects with a different insult, IAA. Reductions to the B and C rings were found to increase the neuroprotective potency of these compounds, but they do not impart enough changes to the molecular structure such that is will not bind to the estrogen receptors. Better yet, the best compounds are potent neuroprotectants, potent antioxidants, and exhibit no binding affinity for estrogen receptor. In our studies, the molecules that fit this description are the di-substituted A-ring steroids, ZYC22, ZYC25, and ZYC26. Improvements may still be possible. One consideration would be to make reductions to the steroid rings of ZYC22, ZYC25, and ZYC26. Five compounds, to date, have been tested in a rat model of middle cerebral artery occlusion. ZYC3, ZYC5, ZYC13, and ZYC26 have proven to be neuroprotective against this model of stroke while ZYC23, the O-methyl congener, was ineffective in this paradigm. 402,403 This lends credence to estrogen’s neuroprotective actions relating to its antioxidant properties and not its ability to bind estrogen receptors. Further, uterotrophic assays showing an increase in weight with estrone but not ZYC26 or ZYC23 substantiate this finding. SAR relationships using cell lines with endogenous ERs or cells with

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87 ectopically expressed ER, ER, or the combination could also be performed to further dissassociate ER involvement. Estratriene protection against other insult models, such as MPTP model for Parkinson’s disease and traumatic brain injury, should also be expected. The mitochondria play major roles in neurodegenerative disease, aging and injury of the brain. There are distinct differences in mitochondrial generation of hydrogen peroxide and levels of antioxidant enzymes differ in males and females. Female animals subjected to ovariectomy possess mitochondria whose activity resembles those of males and E2 replacement reverses this effect. As such, it would be interesting to determine estratriene action on mitochondrial function. 404 Also, it would be interesting to induce lipid peroxidation on isolated mitochondria. Using a fluorescence resonance energy transfer (FRET) assay that measures the mitochondrial function, there was a positive correlation between estratriene neuroprotection and mitochondrial calcium homeostasis. As mitochondria membrane integrity is a major player in apoptosis, its health is fundamental. Further studies are aimed at elucidating the relationship between estrogens and the sulfhydryl status of proteins. Previous work has shown a synergistic interaction between E2 and GSH for neuroprotection. 208 The glutathione system is indispensible for cell protection. Estradiol at 100 nM caused significant increases in glutathione levels in HT-22, primary hippocampal, and primary neocortial cells, but higher concentrations elicited a 76% decrease in basal levels. 405 However, estrogens may interact with proteins with –SH groups as well as small peptides (mercaptans) such as GSH. Oxidative modifications to key proteins (enzymes), such as NMDA receptors, 406 and tau, 407 is a response to cell stress and in some cases is a method devised to affect signaling pathways. Oxidation or

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88 reduction of protein sulfhydryls could diminish or augment activity, promote protein complex formation, or release inhibitory subunits. E2’s ability to interact with sulfhydryl-containing (redox sensitive cysteine groups) proteins, especially those with sulfhydryls in their active domains, such as Na/K ATPase, 408 ryanodine receptors, 409 NMDA receptors, Keap1, 410 and GAPDH 411 will be an area worth exploring. For example, the NMDA receptor channel complex has an extracellular redox site 412 and there are reports that E2 interacts directly with the NMDA receptor. 413 Further, NMDA redox site modulates long-term potentiation (LTP) of NMDA receptors, 414 and estrogens are known to affect LTP. 415,416 Other associations could be made as estrogens have been shown to bind other proteins, estrogen binding proteins, which include GAPDH, OSCP (subunit of ATPase/ATP synthase), -tubulin, 340 and protein disulfide isomerase. 417 Estradiol has also been shown to induce protein thiol/disulfide oxidoreductases (which include PDI, thioredoxin, and glutaredoxin), 300 although estradiol has been shown to inhibit PDI activity by other. 418 PDI upregulation is associated with resistance to ischemic damage. 419 Many signaling pathways are redox sensitive and they include, AP-1, CREB, Erk, HIF-1, NFB, JNK1/SAPK, PKB, PKC, to name a few (for review, see Allen and Tresini). 420 Since estrogens affect many of these redox sensitive pathways, it would be of interest to find the relationship between estrogens and the key regulators in these pathways. For example, estrogens are known to activate MAPK, NFB, and PKB/Akt pathways. The antioxidant response element (ARE) pathway could be a possible mechanism for estratriene neuroprotection. The ARE is a cis-acting regulatory element found in

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89 protective genes such as NAD(P)H;quinone oxidoreductase 1 (QR), glutathione-S-transferase (GSTM3), metallothionein (MT), apoA-I glutathione reductase, GCLR, thioredoxin (TR), and heme oxygenase-1 (HO-1). 213,421,422 Nrf2 is normally found in the cytosol bound to repressor/chaperone, Keap1. Keap1 has a redox sensitive cysteine group that is oxidized by inducers/stressors which makes it dissociate from Nrf2. 423 Unbound Nrf-2 is able to migrate to the nucleus where it then heterodimerically (with Maf2 or other transcription factors) binds to the ARE and stimulates transcription. Stimulators for the ARE include redox-cycling phenols, electrophiles (DMF, H2O2, menadione), phenolic antioxidants, Michael acceptors, xenobiotics, UV light, heavy metals, and oxidants. It seems strange that both oxidative stressors as well as oxidative stress reducers initiate the activity of this response element. However, antioxidants and xenobiotics, alike, are metabolized by enzymes to generate an initial rise in superoxides and electrophiles. It is this initial, small rise in reactive oxygen species (ROS), acting as a preconditioning stimulus, which is then responsible for the activation of the protective genes for cell survival. These low concentrations of ROS modulate physiological processes, which are beneficial and function in intracellular signaling and defense against microorganisms. It is the higher levels of ROS that play a role in aging and neurodegeneration. 424,425 Preliminary evidence (data not shown) found that tert-butylhydroquinone (tBHQ), an antioxidant phenol and potent electrophile activator of the ARE, as well its parent compound butylated hydroxyanisole (BHA) can protect HT-22 hippocampal and C6 glioma cells lines from glutamate-induced oxidative stress in a dose-dependent manner. Further a pulse pretreatment for as little as thirty minutes also enhanced cell survival.

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90 Pretreatment with tBHQ protected human SK-N-SH neuroblastoma cells from the toxic superoxide producing compound menadione. tBHQ also greatly reduces reactive oxygen species (ROS) levels induced by glucose oxidase or the direct addition of hydrogen peroxide using the DCF assay. Furthermore, basal levels of ROS were decreased when treated with tBHQ. However, addition of tBHQ 1h after the addition of OS-generators did not decrease the high levels of ROS. In C6 and SK-N-SH cells, quinone reductase activity was increased with tBHQ treatment. Interestingly, QR expression has been upregulated by antiestrogen-liganded estrogen receptor. Estratrienes could possibly affect ARE action and reporter gene assays would test this hypothesis. The ARE response has been noted in neurons and glia. While glia are usually perceived to play supporting roles in the CNS, it is becoming increasing apparent that glial cells are active participants in brain protection. The sheer number of glia and the battery of antioxidant defense systems found therein make these “supporting” cells important. Compared to neurons, glial cells are more resistant to oxidative stressors. This may be due to more sensitive antioxidant defense mechanisms, including the squelching of ROS, the production of antioxidants (glutathione or cysteine), and the upregulation of antioxidant enzymes. The distribution of reduced GSH was heavily distributed in glial cells of the CNS. 426 In gerbils, hemeoxygenase-1 and HSP25/27 were seen to upregulate in hippocampal and striatal glial cells subjected to global ischemia. 125 Glial conditioned media (GCM) enables the survival and differentiation (neurite growth) of neuronal cells. 427-430

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91 Astroglia are targets for hormones (estrogens and testosterone). These sex hormones play in a role in GFAP expression, growth of astrocytic processes and the extent to which neuronal membranes are covered by astroglial processes. 431 They may also participate with steroids by releasing neuroactive substances and regulating the concentration of growth factors such as IGF-I. The protective actions of hormones during brain injury or peripheral nerve axotomy may be mediated by astrocytes. Estrogens have protected glial cells lines from serum deprivation. 432 Astrocytes normally do not express aromatase in adults but in times of stress will upregulate this enzyme’s actions. Other glial cells, microglia, secrete free radicals as well as cytokines and thus elicit inflammatory responses. Estrogens are known to influence neuroinflammation. Estratriene mediation of microglial-originating toxins would be of interest. In closing, estratrienes with bulky groups flanking the 3-hydroxyl group in the phenolic A ring of the estradiol steroid have proved to be potent neuroprotectants via their antioxidant properties and exhibited negligible estrogen receptor binding. These molecules could be therapeutically effective in oxidative stress injuries without further concern for tumorigenicity and feminizing effects.

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APPENDIX A STRUCTURES ZYC5 ZYC1 ZYC6 ZYC2 ZYC7 ZYC3 ZYC9 ZYC4 ZYC10 92

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93 ZYC11 ZYC16 ZYC12 ZYC17 ZYC13 ZYC18 ZYC14 ZYC19 ZYC15 ZYC20

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94 ZYC21 ZYC26 ZYC27 ZYC22 ZYC23 ZYC28 ZYC24 ZYC29 ZYC25 ZYC30

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95 ZYC33 ZYC38 ZYC34 ZYC39 ZYC35 ZYC40 ZYC36 ZYC41 ZYC37 ZYC42

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96 ZYC43 ZYC48 ZYC44 ZYC49 ZYC45 ZYC50 ZYC46 ZYC51 ZYC52 ZYC47

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97 ZYC53 ZYC54 ZYC55 ZYC56 ZYC57

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98 E2550 E380 E2555 E400 E430 E1240 E2540

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99 PS1 PS2 PS3 PS4

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100 Ent-E2 17E2 DES 17E2

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APPENDIX B TABLE OF EC 50 (IC 50 ) VALUES Compound Glut (10mM) Glut (20 mM) IAA (20 M) IAA (20 M) ER Binding ER Binding TBARs ZYC1 1.025 1.571 0.253 1.72 3.978 4.058 4.922 ZYC2 9.021 10.00 0.006 8.259 126.75 165.8 16.075 ZYC3 0.159 0.365 0.216 10000 10000 1.126 ZYC4 9.207 17.14 196.2 0.052 43.57 31.41 10.171 ZYC5 0.023 0.121 0.120 0.152 10000 10000 1.391 ZYC6 17.61 13.69 2.708 8.483 1698 10000 17.079 ZYC7 8.582 14.73 2.405 1.729 133.5 242.1 5.332 ZYC9 1.956 1.315 5.020 5.296 480.65 204.2 29.465 ZYC10 0.488 0.851 0.296 0.679 16.32 8.138 3.132 ZYC11 4.627 7.031 0.296 0.679 107.0 64.49 26.115 ZYC12 0.548 1.953 1.290 4.440 73.96 53.35 4.763 ZYC13 0.239 1.173 0.191 0.129 25.30 18.70 9.167 ZYC14 0.215 0.240 0.183 0.382 2263 2461 1.033 ZYC15 0.047 0.803 0.646 1.200 1649 10000 1.495 ZYC16 0.882 0.860 2.250 1.912 10000 10000 3.648 ZYC17 0.244 0.270 0.680 0.828 10000 10000 4.913 ZYC18 0.568 0.921 0.264 1.044 2384.5 10000 2.307 ZYC19 0.184 0.272 0.099 0.302 784.1 10000 2.488 ZYC20 0.132 0.273 0.908 1.134 5089 10000 1.905 ZYC21 0.122 0.245 0.108 0.282 2855 4456 1.264 ZYC22 0.030 .030 0.084 0.358 10000 10000 1.827 ZYC23 np np np np 10000 10000 ni ZYC24 0.665 0.768 1.132 3.928 209.45 259.95 7.028 ZYC25 0.238 0.147 0.159 0.359 10000 10000 1.375 ZYC26 0.012 0.037 0.164 0.280 10000 10000 0.708 ZYC27 0.992 1.195 0.541 1.932 14.28 11.97 3.279 ZYC28 0.497 0.568 0.458 1.268 61.12 533.9 7.88 ZYC29 6.730 8.252 1.576 7.525 10.97 17.42 10.47 ZYC30 8.290 9.033 0.591 8.814 26.95 16.84 14.89 ZYC33 0.0938 0.1273 0.046 0.086 10000 10000 1.208 ZYC34 0.121 0.19 0.046 0.219 1460 88.01 0.664 ZYC35 3.203 3.896 1.021 1.623 5.53 14.31 7.806 ZYC36 6.883 6.627 1.195 5.55 2.64 17.445 ZYC37 11.75 8.228 1.836 4.25 4.88 7.48 11.024 np (no protection); ni (no inhibition); 10000 (over 10M required for binding, or no binding activity) 101

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102 Compound Glut (10mM) Glut (20 mM) IAA (20 M) IAA (20 M) ER Binding ER Binding TBARs ZYC38 0.133 0.213 0.021 0.035 296.4 10000 0.895 ZYC39 0.180 0.368 0.049 0.282 633.2 10000 1.577 ZYC40 0.524 0.443 1.901 ZYC41 11.30 1.573 1.341 34.27 1050 6.291 ZYC42 1.167 1.510 0.051 0.942 248.5 323.6 21.89 ZYC43 0.730 1.511 1.393 1.449 8729 7938 4.506 ZYC44 0.363 0.399 0.051 0.167 2637.5 10000 0.408 ZYC45 0.684 0.975 0.239 0.446 800.2 5176 2.214 ZYC46 0.866 0.465 0.069 0.155 7305 10000 2.130 ZYC47 0.139 0.121 0.017 0.088 8831 10000 1.415 ZYC48 0.179 0.527 0.176 1.599 877.5 5208 3.120 ZYC49 np np np np 10000 10000 ni ZYC50 0.111 0.139 0.081 0.235 202.7 769.2 2.156 ZYC51 0.653 1.769 0.446 0.651 1649 4702 1.521 ZYC52 2.439 4.101 0.057 0.286 4258 1143 16.59 ZYC53 14.68 143.2 0.072 0.375 10000 10000 ni ZYC54 5.138 7.621 0.210 0.918 10000 10000 1.580 ZYC55 0.913 0.926 0.246 0.458 10000 10000 1.369 ZYC56 3.550 4.59 0.313 1.717 445.3 354.1 18.60 ZYC57 np np 0.165 1.311 593.9 10000 20.19 ZYC58 1.655 2.922 0.163 0.829 10000 10000 2.564 ZYC59 0.765 0.924 0.162 0.337 752.6 263.9 7.398 E2 3.102 5.739 3.375 161.7 16.46 83.75 E2 1.364 1.978 2.902 4.704 3.041 4.512 19.83 DES 3.772 14.10 0.769 3.802 2.615 1.84 6.882 E1 3.029 8.920 2.092 1609.2 13.35 30.92 80.79 EntE2 0.936 1.166 7.110 35.01 24.65 5.635 23.95 E380 1.277 1.980 0.159 1.230 5.912 2.372 6.725 E400 1.651 1.866 0.126 1.245 117.5 52.37 20.88 E430 np np 7.923 33.47 1880 10000 ni E1240 np np np np 785.5 1197 ni E2540 np np np np ni E2550 np np np np 953.4 260.3 ni E2555 np np np np 1513 10000 ni E2560 np np np np 33.06 261.7 ni PS1 13.08 13.84 17.45 2.547 29.04 12.22 9.701 PS2 10.15 16.21 278.7 1662 27.65 33.09 8.951 PS3 np np np np 692.2 615.4 ni PS4 np np np np 2862 10000 ni np (no protection); ni (no inhibition); 10000 (over 10M required for binding, or no binding activity)

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APPENDIX C SPEARMAN’S RANK CORRELATION Glut (20) IAA (20) IAA (40) ER ER TBAR Glut (10) 0.9218 1.042 <0.0001 0.3567 1.674 <0.0001 0.4861 1.434 <0.0001 0.1535 -2.552 0.0015 0.1269 -2.807 0.0042 0.6637 1.227 <0.0001 Glut (20) 0.3930 1.595 <0.0001 0.4937 1.423 <0.0001 0.1894 -2.298 0.0004 0.1600 -2.500 0.0012 0.6483 1.242 <0.0001 IAA (20) 0.6008 1.2900 <0.0001 0.06943 -3.795 0.0369 0.08524 -3.425 0.0202 0.3315 1.737 <0.0001 IAA (40) 0.1452 -2.625 0.0021 0.1471 -2.607 0.0019 0.5063 1.405 <0.0001 ER 0.8485 1.086 <0.0001 0.2047 -2.210 0.0020 r s 1/slope ER P value 0.1717 -2.413 0.0007 103

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BIOGRAPHICAL SKETCH Evelyn Perez was born in Jacksonville, Florida, in 1974. She is the middle child of Jasmin and Romer Perez and the sibling to Joy and Jesse Ronald Perez. As a child of a navy parent, she was able to spend her younger years in Virginia, Texas, and California. Her father retired to Jacksonville, Florida, where she graduated from high school and attended undergraduate school. She finished her biology degree at the end of 1996 and then pursued an advanced degree with the Department of Pharmacodynamics at the University of Florida in Gainesville, Florida, which was a little more than an hour away from home. As graduate life was settling down, her boss decided to move to Fort Worth, Texas, with six of his laboratory personnel. Three and a half years later, she is thankful for the opportunity to make wonderful friendships and working relationships in two different places. She is looking forward to being happy in her career and home life. 152