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Characterization of the Inhibition of Genistein

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Characterization of the Inhibition of Genistein CHARACTERIZATION OF THE INHIBITION OF GENISTEIN GLUCURONIDATION BY BISPHENOL A IN HUMAN AND RAT LIVER MICROSOMES By JANIS LAURA COUGHLIN A thesis submitted to the Graduate School-New Brunswick Rutgers, The State University of New Jersey and The Graduate School of Biomedical Sciences University of Medicine and Dentistry of New Jersey in partial fulfillment of the requirements for the degree of Master of Science Joint Graduate Program in Toxicology written under the direction of Dr. Brian Buckley and approved by __________________________________ __________________________________ __________________________________ __________________________________ New Brunswick, New Jersey OCTOBER 2011 ABSTRACT OF THE THESIS Characterization of the Inhibition of Genistein Glucuronidation by Bisphenol A in Human and Rat Liver Microsomes By JANIS LAURA COUGHLIN Thesis Director: Dr. Brian Buckley Genistein is a natural phytoestrogen that is found abundantly in the soybean. Bisphenol A (BPA) is a synthetic chemical used in the synthesis of polycarbonate plastics and epoxy resins. Endocrine disrupting properties of both genistein and BPA have been well established by various laboratories. Because the adverse biological effects caused by genistein and BPA are similar, and may include common co-exposure scenarios in the general population such as in the consumption of a soy-milk latte from a polycarbonate plastic coffee mug, analysis of the perturbation of the metabolism via glucuronidation of genistein in the presence of BPA has been assessed. Human and rat liver microsomes were exposed to varying doses of genistein (0 to 250 μM) in the absence (0 μM) or presence (25 μM) of BPA. Treatment with 25 μM BPA caused non-competitive inhibition of the glucuronidation of genistein in human liver microsomes with a Ki value of 58.7 μM, represented by a decrease in Vmax from 0.93 ± 0.10 nmol/min/mg in the ii absence of BPA to 0.62 ± 0.05 nmol/min/mg in the presence of BPA, and a negligible change in Km values between treatment groups. The addition of BPA to incubations performed in rat liver microsomes resulted in competitive inhibition of the glucuronidation of genistein at a Ki of 35.7 μM; Vmax values remained steady (2.91 ± 0.26 nmol/min/mg in the absence of BPA and 3.05 ± 0.41 nmol/min/mg in the presence of BPA), while Km values increased in the presence of BPA (49.4 ± 14 μM in the absence of BPA and 84.0 ± 28 μM in the presence of BPA). These findings indicate that the type of inhibition on genistein glucuronidation exerted by BPA differs among species. iii ACKNOWLEDGEMENTS I would like to thank a handful of people who have supported me in various ways throughout my studies and research. Many thanks to my mentor Dr. Brian Buckley, who graciously welcomed me into his lab with open arms. I am incredibly fortunate to have had the opportunity to work for such a wonderful advisor. He has taught me so much, both professionally and personally, and has always greeted me with a smile. I sincerely appreciate that he always made time for my questions and concerns, regardless of how unimportant or trivial they may have been. I am incredibly grateful for his unparalleled mentoring and guidance. Special thanks are extended to my committee members, Dr. Kenneth Reuhl, Dr. Lauren Aleksunes, and especially Dr. Paul Thomas for his tremendous patience and the countless hours he spent with me designing experiments, checking calculations, and discussing and analyzing results of the metabolism experiments. I truly appreciate him opening his lab to me so freely, and for being such a great example of not only a phenomenal scientist, but also a wonderful person. A big “thank you” to the past and present members of the Buckley lab, especially Dr. Bozena Winnik for her tireless assistance in the method development, Dr. Hilly Yang for his technical assistance and troubleshooting of the HPLC-MS/MS, and Dr. Elizabeth McCandlish for her chemistry expertise. I greatly appreciate the financial support provided by the National Institutes of Health (Grant Number T32ES007148), National Institute of Environmental Health iv Sciences (Grant Number ES05022), and the Environmental and Occupational Health Sciences Institute. A special thank you to Michal Sheleg, who somehow made studying fun, and always made lunchtime quite cheerful! A big mahalo to my parents, who have generously supported me in so many ways. I have no idea how to even begin to repay them. Merci un mille fois to Stu, whose unwavering support and unfaltering belief in me gave me the courage to follow my heart, and had made all the difference in my life. Heaven only knows where I would be without him. v TABLE OF CONTENTS Page ABSTRACT OF THE THESIS…………………………………………... ii ACKNOWLEDGEMENTS…………………………………………...….. iv LIST OF TABLES………………………………………………………… ix LIST OF FIGURES……………………………………………………….. x LIST OF ABBREVIATIONS…………………………………………….. xi 1.0. GENERAL INTRODUCTION………………………………………. 1 1.1. Endocrine Disrupting Compounds...…………………………. 1 1.2. Glucuronidation………………………………………………. 3 1.2.1. Enzyme Inhibition………………………………….. 4 1.3. Genistein……………………………………………………… 7 1.3.1. Affinity for the estrogen receptor……….…….…… 8 1.3.2. Biological effects………………………………...…. 8 1.3.3. Pharmacodynamics and pharmacokinetics……...….. 11 1.3.4. Environmentally relevant exposure levels……..…… 12 1.4. Bisphenol A…………………………………………..………. 14 1.5. Rationale for Analysis of Genistein and BPA………………… 17 1.5.1 Comparable metabolism…………………………….. 17 1.5.2. Similar biological effects…………………………… 18 1.5.3. Common exposures…………………………………. 19 2.0. AIM ONE: DETERMINATION VIA SPE AND HPLC-MS/MS…... 20 2.1. Abstract………………………………………………...…….. 20 vi 2.2. Introduction…………………………………………….……. 21 2.3. Materials and Methods………………………………….…… 25 2.3.1. Solid phase extraction……………….……..……… 26 2.3.2. Standard solutions…………………….…………… 26 2.3.3. Blank controls………………………….………….. 27 2.3.4. Chromatographic conditions………………………. 27 2.3.5. Mass spectrometry…………………………………. 28 2.3.6. Statistical analyses…………………………………. 29 2.4. Results………………………………………………...……... 30 2.4.1. Retention times, limits of detection, and calibration curves……………………………………………….. 30 2.4.2. Recoveries………………………………………….. 30 2.4.2.1. Bond Elut Plexa SPE cartridges………….. 31 2.4.2.2. Oasis HLB SPE cartridges………………... 31 2.4.2.3. UCT C18 SPE cartridges…………………. 31 2.4.2.4. Glucuronide recoveries……………...……. 32 2.4.3. HPLC column performance………………………… 32 2.5. Discussion…………………………………………………….. 34 2.6. Conclusion……………………………………………………. 37 3.0. AIM TWO: INHIBITION OF GLUCURONIDATION…………….. 38 3.1. Abstract………………………………………………...…….. 38 3.2. Introduction…………………………………………….……. 40 3.3. Materials and Methods………………………………….…… 43 vii 3.3.1. Glucuronide formation……………….……..……… 43 3.3.2. Inhibition studies…………………..….…………… 44 3.3.3. Sample preparation...…………………….…..…….. 44 3.3.4. Standard solutions…………………………………. 45 3.3.5. Blank controls…..…………………………………. 45 3.3.6. Analytical conditions………………………………. 45 3.3.7. Data analyses………………………………………. 46 3.4. Results………………………………………………...……... 49 3.4.1. BPA glucuronidation kinetics……………………… 49 3.4.2. Genistein glucuronidation kinetics……………..….. 49 3.5. Discussion…………………………………………………….. 51 3.6. Conclusion……………………………………………………. 54 4.0. GENERAL DISCUSSION…………………………………………… 55 4.1. Areas of further research……………………………………… 59 5.0. REFERENCES………………………………………………………... 61 viii LIST OF TABLES 1.1. Estrogenic potentials of 17 β-estradiol, genistein, and BPA………………... 70 2.1. Summary of mass spectrometric parameters………………………………… 71 2.2. Retention times of analytes using Kinetex C18 and Discovery C8..………… 72 ix LIST OF FIGURES 1.1. Chemical structures of genistein and genistein gluc…………….…………… 73 1.2. Chemical structure of 17 β-estradiol…………………………………………. 74 1.3. Chemical structures of BPA and BPA gluc………..…………….…………… 75 1.4. Genistein and BPA co-exposure scenario……………………………………. 76 2.1. Chromatograms and mass spectra using Kinetex C18………………………. 77 2.2. Chromatograms and mass spectra using Discovery C8…..…………………. 78 2.3. SPE recoveries of BPA and genistein………………………………….…….. 79 2.4. Recoveries of analytes from optimized SPE conditions……………………… 80 3.1. Optimization of HLM protein concentration………………………………… 81 3.2. Optimization of RLM protein concentration………………………………… 82 3.3. BPA glucuronidation kinetics………………………………………………… 83 3.4. Genistein glucuronidation kinetics in HLMs, pooled data……………………. 84 3.5. Genistein glucuronidation kinetics in RLMs, pooled data……………………. 85 3.6. IC50 of BPA for genistein glucuronidation in HLMs ……………………….. 86 3.7. Genistein glucuronidation kinetics in HLMs, replicate 1 ……………………. 87 3.8. Genistein glucuronidation kinetics in HLMs, replicate 2 ……………………. 88 3.9. Genistein glucuronidation kinetics in HLMs, replicate 3 ……………………. 89 3.10. Genistein glucuronidation kinetics in RLMs, replicate 1 …………….……. 90 3.11. Genistein glucuronidation kinetics in RLMs, replicate 2 ………….………. 91 3.12. Genistein glucuronidation kinetics in RLMs, replicate 3 ……………..……. 92 x LIST OF ABBREVIATIONS BPA bisphenol A BPA gluc bisphenol A β-D-glucuronide DES diethylstilbestrol EDC endocrine disrupting compound EA 3 mL ethyl acetate and 3 mL acetonitrile ER estrogen receptor EM 3 mL ethyl acetate and 3 mL methanol EMA 3 mL ethyl acetate, 2 ½ mL methanol, and 2 ½ mL acetonitrile ESI electrospray ionization genistein gluc genistein 4’-β-D-glucuronide HLB hydrophilic-lipophilic balance HLM human liver microsomes HPLC high performance liquid chromatography HPLC-MS/MS high performance liquid chromatography tandem mass spectrometry MA 3 mL methanol and 3 mL acetonitrile
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