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Proquest Dissertations INFORMATION TO USERS This manuscript has been reproduced from the microfilm master. UMI films the text directly from the original or copy submitted. Thus, some thesis and dissertation copies are in typewriter tace, while others may be from any type of computer printer. The quality of this reproduction is dependent upon the quality of the copy submitted. Broken or indistinct print, colored or poor quality illustrations and photographs, print bleedthrough, substandard margins, and improper alignment can adversely affect reproduction. In the unlikely event that the author did rrot send UMI a complete manuscript and there are missing pages, these will be noted. Also, if unauthorized copyright material had to be removed, a note will indicate the deletion. Oversize materials (e.g., maps, drawings, charts) are reproduced by sectioning the original, t)eginning at the upper left-hand comer and continuing from left to right in equal sections with small overlaps. Photographs included in the original manuscript have t>een reproduced xerographically in this copy. Higher quality 6* x 9" black and white photographic prints are available for any photographs or illustrations appearing in this copy for an additional charge. Contact UMI directly to order. Bell & Howell Information and Leaming 300 North Zeeb Road, Ann Arbor, Ml 48106-1346 USA 800-521-0600 UMT OXIDATIVE MECHANISMS OF ESTROGEN INDUCED CARCINOGENESIS DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By By James Austin Mobley ***** The Ohio State University 2000 Dissertation Committee: Approved by Robert W. Brueggemeier, Adviser ^ Pui-Kai Li Young C. Lin Robert W. Brueggemeier Larry W. Robertson Adviser College of Pharmacy UMI Number 9971609 UMI UMI Microform9971609 Copyright 2000 by Bell & Howell Information and Leaming Company. All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code. Bell & Howell Information and Leaming Company 300 North Zeeb Road P.O. Box 1346 Ann Aitor, Ml 48106-1346 ABSTRACT The growth promoting effects of estrogens in hormone-dependent tumor tissues involve receptor-mediated pathways that are well recognized; however, the role of estrogens in tumor initiation remains controversial. Estrogen metabolites, primarily the catechol estrogens (CE’s), have been implicated in tumor initiation via a redox cycling mechanism. Reports regarding estrogen mediated DNA damage include the induction of 8-oxo-2'-deoxyguanosine (8-oxo-dG) in vitro and in vivo, indicating a role for oxidative stress in tumor initiation and/ or progression. Estrogens also appear to play a role in modulating the redox state of endothelial, neuronal, and gynecological related tissues, behaving as antioxidants in some tissues and pro-oxidants in others. We have developed metabolically stable CE analogs for the study of receptor vs. redox cycling effects on DNA damage. Comparisons between hydroxy estradiols (OH- Ei's), and hydroxymethyl estradiols (HMEz) in potentiometric and DNA damaging studies were made. DNA damage was assessed in calf thymus DNA using 8-oxo-2’- deoxyguanosine (8-oxo-dG) as a genotoxic marker for oxidative stress. Increases in 8- oxo-dG/10® dG were significant for each Z-OH-Ez and 4-OH-Ez Cu(II)S 0 4 , a transition metal known to catalyze the redox cycling of o-quinones, substantially increased DNA damage by both CE’s. However, DNA damage was only observed at concentrations of 10 fiM or higher, much greater than what is found under physiologic conditions. Furthermore the presence of endogenous antioxidants such as glutathione, SOD, and catalase drastically reduced DNA damage induced by high concentrations of Z-OH-Ez. Thus, both 2 -OH-E2 and 4 -OH-E2 are capable of producing oxidative DNA damage at micromolar concentrations in vitro; however, based on data herein, it is unlikely that free radical production via redox cycling of CE's is a causative factor in human tumorigenesis. There was no DNA damage observed for the non-redox cycling HMEz's, making these compounds useful probes in the study of receptor-mediated carcinogenesis. DNA isolation, cellular DNA repair and high antioxidant status have made the measurement of 8-oxo-dG in vivo and in cell culture very challenging. Of methods utilized to potentiate DNA damage in cells, GSH depletion through specific inhibition of the y-glutamylcysteine transferase enzyme using buthionine sulphoximine (BSC) has shown significant promise. Depletion of GSH in the ER-positive MCF-7 cell line resulted in a maximal induction of 8-oxo-dG/ 10^ dG by 30 minutes following the addition of 500 |iM H2O2, nearly returning to baseline by 2 hours with no appreciable increase in cell death. Similarly, the addition of H 2O2 for 30 minutes resulted in an increase in 8-oxo-dG/ 10^ dG formation in GSH depleted vs. non-depleted cells from 115 % to 183 % respectively. Treatment of GSH depleted cells with 10 pM 2-OH-E2 and 4-OH-E2 resulted in an increase in 8-oxo-dG/ 10* dG of 127 % and 160 % respectively. There was a significant potentiation in CE induced DNA damage with the addition of Cu(Il) for both the 2- and 4-CE’s of 165 % and 200 % respectively. Ill Interestingly, the treatment of GSH depleted MCF-7 cells with 100 nM and 1.0 |iM estradiol alone for only 18 hours increased DNA damage by 145 % and 189 % respectively. BSO has been utilized as a means of increasing sensitivity to oxidative DNA damage without causing a marked increase in baseline 8-oxo-dG or increase in cell death. This model was developed in order to study the role of estrogens in the breast cancer initiation process. Treatment of MCF-7 cells with BSO and 10 nM Ez resulted in an increase in sensitivity to peroxide-induced oxidative DNA damage as measured by an increase in 8- oxo-dG/ 10^ dG. Treatment of MCF-7 cells with 10 nM estradiol alone resulted in a decrease in antioxidant capacity as measured by peroxide metabolism that was paralleled by a decrease in catalase activity. These effects were all opposed by the antiestrogen tamoxifen. The estrogen-mediated decrease in catalase activity and sensitivity to DNA damage was not observed in MDA-MB-231 cells. Treatments with 10 nM estradiol in MCF-7 cells resulted in increased glutathione peroxidase, superoxide dismutases (I) and (II) and gIucose-6-phosphate dehydrogenase activities, while total glutathione content and catalase activity decreased significantly. This indicates that the antioxidant status of MCF-7 cells can be modulated through the actions of the estrogen receptor. If so, this may explain some of the estrogen induced pro-oxidant effects reported in-vivo at physiologic concentrations. Measurement of catalase activity in synchronized MCF-7 cells treated with 10 nM Ez indicated that the decrease in antioxidant status is cell cycle regulated with a maximal decrease at the beginning of Gi followed by an increase just prior to S phase. Thus, a novel mechanism by which estradiol is capable of down regulating catalase activity through the estrogen receptor, albeit likely indirectly, has IV been reported here. This regulation o f antioxidant status appears to be cell cycle related for the purpose of allowing a modest increase in growth promoting reactive oxygen secondary messengers. We have shown that this estrogen-induced mechanism increases the susceptibility of ER positive cells to oxidative DNA damage at physiologic concentrations and may be the key relating estrogens to breast cancer initiation and/ or progression. A number of assays were developed or simply adapted for future studies involving estrogen metabolism and DNA damage with the goal that the catechol estrogen analogs previously studied will be utilized. Dedicated To My Wife and Best Friend Luz Minerva Burgos Faster de Mobley VI ACKNOWLEDGMENTS • Graduate School: Dr. Brueggemeier, Dr. Robertson, Dr.Lin, Dr. Olson, Dr. Sugimoto, Dr. Darby, Abhijit, Pam, Jack, Nancy, Holly, Jen, La Rae, Trevor, Jeanette, Surachai, Jill, Muriel, Punit, Anne, Carl, Larry, Melanie, Famhas, Anna, Randy, John, Young, Danielle, Xingu. • Roxane Labs: Gary, Ron, John, Christallin, Jennifer, Dave. • Oregon State University: Dr. Ingle, Dr. Thomas, Dr. Nibbler, Dr. Pastorek. • Evanite: Gabriel, Buddy, John, Bob, Rich, Dan, Farooqui. • Other Inspirational Friends: Pat D , Mike C., Phillip, Dave H., Mike H., Brian, Chris T., Mark N., Chris M., Terry J. • Family: John, Brenda, Lisa, Allan, Mom and Dad. Special Thanks • Dr. Minerva Burgos, for putting up with my graduate school induced emotional imbalances, listening to all of my crazy ideas, continued support throughout everything, and finally for feeding me Puerto Rican food whenever I begged for it. VII • My Mother, for giving me independence, for reinforcing creativity, and for making my life goal one o f research in cancer. • My Father, for inspiring me to work hard and continually drive toward something better. • My Brother Allan, for what he would have been if given the chance. • Dr. Robert Brueggemeier, for making graduate school possible, for being a very flexible and understanding adviser, and most importantly for giving me the first step in a lifetime o f research that I hope to endure and enjoy. • Dr. James Ingle, for introducing me to research and making it fun. • Trevor Petrel, for giving me the means by which to travel to work on a daily basis, for making bad research days tolerable, and for keeping me sane during my elongated non-productive period in the lab. • Dr. Ron Shockely, for all of the long discussions over medicine during those early morning runs after work, and for giving me the drive to never stop leaming. • Dr. Larry Cain and Dr. Anderson, for the unselfish act o f loaning me all o f the equipment and advice needed to complete the electrochemical studies. • Jeannette Richards and Abhijit Bhat, for listening to my crazy oxidative stress talk that everyone knows is the foundation of every major disease (just kidding), and especially for taking the time to endure and edit many of my scribbles.
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