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Effect of hypophysectomy on induction of mammary cancer and CYP1 enzymes in Sprague-Dawley rats Leung, Grace S. 2006

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E F F E C T O F H Y P O P H Y S E C T O M Y O N INDUCTION O F M A M M A R Y C A N C E R AND CYP1 E N Z Y M E S IN S P R A G U E - D A W L E Y RATS by G R A C E S. L E U N G B.Sc, The University of British Columbia, 2003 A THESIS SUBMITTED IN PARTIAL F U L F I L L M E N T O F T H E R E Q U I R E M E N T S F O R T H E D E G R E E O F M A S T E R OF SCIENCE in T H E F A C U L T Y O F G R A D U A T E STUDIES (Pharmaceutical Sciences) T H E UNIVERSITY OF BRITISH C O L U M B I A A U G U S T 2006 © Grace S. Leung, 2006 A B S T R A C T Breast cancer is the most prevalent cancer among Canadian women and is the second leading cause of cancer-related deaths in North America. Hormonal influences including early menstruation, reproductive history, and hormone replacement therapy usage strongly affect breast cancer risk and development. In addition, hormone deprivation such as ovariectomy, hypophysectomy, and anti-estrogen therapy have been used as treatments to slow breast cancer growth. In the present study, we determined the effect of hypophysectomy on mammary carcinogenesis. Twenty intact and hypophysectomized (at 7 to 7.5 weeks) virgin Sprague-Dawley rats were treated with a single intragastric administration of 20 mg of 7,12-dimethylbenz[a]anthracene (DMBA) or an equivalent volume of com oil between 50 to 60 days of age. None of the hypophysectomized rats developed mammary tumors at 120 days post-treatment, whereas 55% of intact rats treated with D M B A developed mammary tumors. D M B A is a polyaromatic hydrocarbon procarcinogen that requires metabolic activation by the cytochrome P450 (CYP) system and microsomal epoxide hydrolase (mEH) prior to becoming carcinogenic. To determine if CYP and mEH enzymes needed for D M B A activation are down-regulated in hypophysectomized rats, CYP1A1, CYP1A2, CYP1B1, and mEH protein levels and C Y P 1-mediated enzyme activities were measured in liver and mammary tissue. Immunoblot analysis showed that there were no differences in hepatic CYP1A1 and CYP1A2 levels between D M B A - or corn oil-treated intact and hypophysectomized rats. The results also showed that mammary CYP1 A l , CYP1A2, and CYP IB 1 from hypophysectomized and intact rats were induced by D M B A . Microsomal E H levels in the liver and mammary gland were increased in hypophysectomized rats when ii compared to the intact animals and D M B A treatment did not further affect mEH expression. MROD and BaP hydroxylase activities were similar in corn oil-treated hypophysectomized and intact rats and D M B A treatment increased both activities in hypophysectomized and intact rats to a similar extent. Based on the results of this study, the lack of mammary tumorigenesis in DMBA-treated hypophysectomized rats cannot be ascribed to the inability of either hepatic or mammary tissue to bioactivate D M B A . i n TABLE OF CONTENTS Page Abstract ii Table of Contents iv List of Figures vii List of Tables ix List of Abbreviations x Acknowledgements xi 1 INTRODUCTION 1 1.1 Incidence and Types of Human Breast Cancer 1 1.2 Etiology and Risk Factors of Human Breast Cancer 5 1.3 Carcinogenicity of Estrogens 6 1.4 Hormonal Regulation of Breast Cancer 9 1.4.1 Ovarian hormones and mammary cancer 9 1.4.2 Pituitary factors and mammary cancer 10 1.5 Cytochrome P450 Enzymes Overview 12 1.5.1 C Y P 1 enzymes overview 13 1.6 Epoxide Hydrolase Overview 15 1.7 Animal Model of Human Breast Cancer 16 1.8 Chemical Carcinogen Induction of Mammary Cancer in Rodents 16 1.9 D M B A Overview 18 1.9.1 Bioactivation of D M B A 19 1.9.2 Carcinogenic metabolites of D M B A , 23 1.10 Rationale and Goals of the Present Study : 26 1.11 Experimental Hypotheses 26 1.12 Specific Objectives 27 2 MATERIALS A N D METHODS 28 2.1 Chemicals 28 2.2 Animals 32 2.3 Study Designs 33 2.3.1 Study 1: DMBA-induced mammary tumorigenesis in Sprague-Dawley rats..33 2.3.2 Study 2: Expression and activity of DMBA-bioactivating enzymes in intact and hypophysectomized rats 35 2.4 Microsome Preparation 37 2.4.1 Liver 37 2.4.2 Mammary Tissue 37 2.5 Total C Y P Determination 38 2.6 Total Protein Determination 38 iv 2.7 SDS-PAGE and Immunoblot Assay 39 2.8 Quantitation of Immunoblot Staining 41 2.9 Enzyme Activity 42 2.9.1 Methoxyresorufin 0-demefhylase (MROD) assay 42 2.9.2 Benzo[a]pyrene hydroxylase assay 43 2.10 Statistical Analysis 44 RESULTS 45 3.1 Study 1: DMBA-Induced Mammary Tumorigenesis in Sprague-Dawley rats 45 3.1.1 Effects of hypophysectomy and D M B A treatment on body weight 45 3.1.2 Effects of hypophysectomy and D M B A treatment on organ weight 47 3.1.3 Effect of a single intragastric dose of 20 mg of D M B A on mortality 48 3.1.4 Effect of hypophysectomy on DMBA-induced mammary tumor incidence...49 3.2 Study 3: Expression and Activity of DMBA-bioactivating enzymes in intact and hypophysectomized rats 51 3.2.1 Effects of hypophysectomy and D M B A treatment on body and liver weight.51 3.2.2 Effects of hypophysectomy and D M B A treatment on total C Y P content 52 3.2.3 Effects of hypophysectomy and D M B A treatment on hepatic C Y P 1A2 protein expression 52 3.2.4 Effects of hypophysectomy and D M B A treatment on hepatic CYP 1A1 protein expression 53 3.2.5 Effects of hypophysectomy and D M B A treatment on hepatic CYP1B1 protein expression 56 3.2.6 Effects of hypophysectomy and D M B A treatment on mammary CYP1A2 protein expression 56 3.2.7 Effects of hypophysectomy and D M B A treatment on mammary CYP1 A l protein expression 56 3.2.8 Effects of hypophysectomy and D M B A treatment on mammary CYP1B1 protein expression 57 3.2.9 Effects of hypophysectomy and D M B A treatment on hepatic mEH protein expression 60 3.2.10 Effects of hypophysectomy and D M B A treatment on mammary mEH protein expression 61 3.2.11 Effects of hypophysectomy and D M B A treatment on hepatic enzyme activity 65 3.2.11.1 Validation of MROD assay conditions: Calibration curve 65 3.2.11.2 Validation of MROD assay conditions: Saturating substrate concentration 67 3.2.11.3 Validation of MROD assay conditions: Reaction time 69 3.2.11.4 Effects of hypophysectomy and D M B A treatment on MROD activity..70 3.2.11.5 Validation of BaP hydroxylase assay conditions: Inter-assay variation..71 3.2.11.6 Validation of BaP hydroxylase assay conditions: Reaction time 71 3.2.11.7 Effects of hypophysectomy and D M B A treatment on BaP hydroxylation activity 73 4 DISCUSSION 75 4.1 Effect of Hypophysectomy on Growth and Organ Development 76 4.2 Effect of D M B A Treatment on Mammary Tumorigensis 78 4.3 Effect of Hypophysectomy and D M B A Treatment on Mammary Tumorigenesis...79 4.4 Adverse Effects Associated with D M B A Treatment 80 4.5 Bioactivation of D M B A in Hypophysectomized Rats 82 4.6 Effect of Hypophysectomy on CYP 1 and mEH Enzymes Expression 83 4.7 Effect of D M B A Treatment on CYP1 and mEH Enzyme Expression 86 4.8 Methods to Measure D M B A Bioactivation 87 4.9 Effects of Hypophysectomy and D M B A Treatment on Enzyme Activities 88 4.10 Evaluation of Hormones and DMBA-Induced Mammary Tumorigenesis 92 4.11 Summary 94 4.12 Conclusion and Future Directions 95 5 REFERENCES 97 6 APPENDIX 112 6.1 Appendix I: Mortality in Hypophysectomized Rats Given Intermittent D M B A doses 112 6.1.1 Introduction 112 6.1.2 Experimental design 112 6.1.3 Effect of intermittent D M B A treatment on body weight 113 6.1.4 Effect of intermittent D M B A treatment on mortality 113 vi LIST OF FIGURES Page Figure 1.1 Types of lobule in human mammary gland 2 Figure 1.2 Development of the human mammary gland 4 Figure 1.3 The bioactivation of estrone and estradiol leading to breast cancer 8 Figure 1.4 The chemical structure of D M B A 18 Figure 1.5 Metabolic activation of D M B A by one-electron oxidation and diol epoxide pathways 21 Figure 1.6 The bioactivation pathways of D M B A to its carcinogenic metabolite 22 Figure 1.7 Overview of metabolic activation of D M B A leading to cancer 24 Figure 1.8 Formation of stable and depurinating D N A adducts and the generation of apurinic sites 25 Figure 2.1 Treatment plan for study 1: DMBA-induced mammary tumorigenesis in Sprague-Dawley rats 34 Figure 2.2 Treatment plan for study 2: Expression and activity of D M B A -bioactivating enzymes in intact and hypophysectomized rats 36 Figure 2.3 The cytochrome P450-catalyzed demethylation of methoxyresorufin 43 Figure 2.4 The cytochrome P450-catalyzed hydroxylation of benzo[a]pyrene 44 Figure 3.1 Daily mean body weight of rats in each treatment group during the experimental period of study 1 46 Figure 3.2 Total number of palpable mammary tumors in each group post D M B A or corn oil treatment 50 Figure 3.3 Immunoblot showing hepatic samples probed with rabbit anti-rat CYP1A2 polyclonal serum 54 Figure 3.4 Summary of hepatic CYP1A1 and CYP1A2 protein content in corn oil-or DMBA-treated hypophysectomized and intact rats 55 vii Figure 3.5 Immunoblot showing mammary samples probed with rabbit anti-rat CYP1A2 polyclonal serum 58 Figure 3.6 Immunoblot showing mammary samples probed with rabbit anti-rat CYP1B1 antibody 59 Figure 3.7 Immunoblot showing hepatic samples probed with rabbit anti-rat mEH IgG 62 Figure 3.8 Immunoblot showing mammary samples probed with rabbit anti-rat mEH IgG 63 Figure 3.9 Summary of hepatic and mammary E H protein content in corn oil- or DMBA-treated hypophysectomized and intact rats 64 Figure 3.10 Calibration curve for the MROD assay 66 Figure 3.11 Effect of varying substrate concentrations on resorufin formation in hepatic microsomes 68 Figure 3.12 The formation of resorufin over time from 0 to 16 minutes in hepatic microsomes 69 Figure 3.13 The formation of OH-BaP over time from 0 to 12 minutes in hepatic microsomes 72 Figure 4.1 The hypothalamus interacts with the pituitary gland through secretion of gonadotropin releasing hormone 93 Figure 6.1 Experimental design: Mortality in hypophysectomized rats given intermittent D M B A doses 113 Figure 6.2 Average body weights of rats during the experimental period 114 viii LISTS O F T A B L E S Page Table 2.1 Buffers and reagents used in this study 31 Table 2.2 Amount of microsomal protein loaded per lane for immunoblot assay 40 Table 2.3 Concentration of antibody used for immunoblot assay 40 Table 3.1 Effects of hypophysectomy and D M B A treatment on final mean body weight 47 Table 3.2 Effects of hypophysectomy and D M B A treatment on final mean organs weight 48 Table 3.3 Number of rats in each group before treatment and at termination 48 Table 3.4 Mammary tumor incidence, weight, multiplicity, and latency in intact and hypophysectomized rats following treatment with D M B A or corn oil 49 Table 3.5 Effects of hypophysectomy and D M B A treatment on body weight and liver weight in female rats terminated 24 hours post treatment 51 Table 3.6 Effects of hypophysectomy and D M B A treatment on total hepatic CYP content 52 Table 3.7 Effects of hypophysectomy and D M B A treatment on hepatic microsomal M R O D activities 71 Table 3.8 Effects of hypophysectomy and D M B A treatment on hepatic microsomal BaP hydroxylase activities 74 i x LIST O F ABBREVIATIONS A C T H adrenocorticotrophic hormone AhR aromatic hydrocarbon receptor BaP benzo[a]pyrene BCIP 5-bromo-4-chloro-3-indoyl phosphate,/?-toluidine salt cDNA complementary deoxyribonucleic acid CO corn oil CYP cytochrome P450 D M B A 7,12-dimethylbenz[a]anthracene D N A deoxyribonucleic acid DRE dioxin response element Ei estrone E 2 estradiol mEH microsomal epoxide hydrolase FSH ollicule-stimulating hormone GH growth hormone HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid Hypox hypophysectomized L H luteinizing hormone i.g. intragastric IgG immunoglobulin G 3-MC 3-methylcholanthrene mA milliamp mg milligram ml milliliter M N U /V-methyl-/V-nitrosourea mRNA messenger ribonucleic acid MROD methoxyresorufin O-demethylase N A D P H nicotindiamide adenine dinucleotide phosphate tetrasodium salt NBT nitro blue tetrazolium PAH polycyclic aromatic hydrocarbon PBS phosphate buffered saline PCDD polychlorinated dibenzo-p-dioxin pmol picomole PRL prolactin RT-PCR reverse transcription polymerase chain reaction RNA ribonucleic acid SDS sodium dodecyl sulphate TCDD 2,3,7,8-tetrachlorodibenzo-/?-dioxin TEMED N,N,N ' N '-tetramethylenediamine mg milligram ul microliter X R E xenobiotic response element x A C K N O W L E D G E M E N T S Thank you to my supervisors, Dr. Stelvio Bandiera and Dr. Thomas Chang, for their support, help, and understanding throughout my research and their guidance in writing the thesis. I would also like to thank Jenny Tai and Dr. Eugene Hrycay as well as other lab members for the assistance of my experiments. I would also like to acknowledge Canadian Breast Cancer Research Alliance for the funding of my project, Merck Research Laboratories for the graduate training scholarship, and University of British Columbia for the teaching assistantship. Special thanks to my parents, whose have supported me throughout my years of education, both morally and financially. xi 1. INTRODUCTION 1.1 Incidence and Types of Human Breast Cancer Breast cancer is the most common cancer among women and is the second leading cause of cancer-related death in North America. According to Canadian Cancer Statistics, it is estimated that 21,600 women were newly diagnosed with breast cancer in 2005 and there were 5,300 deaths from the disease (Canadian Cancer Statistics, 2005). On average, one in 9 women is expected to develop breast cancer during her lifetime. Breast cancer can also affect men. An estimated 150 men were newly diagnosed and 45 deaths were associated with breast cancer in 2005 (Canadian Cancer Statistics, 2005). Although breast cancer mortality rates among women over the past three decades have declined slightly, breast cancer continues to be the leading type of cancer among Canadian women. Adenocarcinoma is a type of carcinoma that arises from epithelial gland cells. Most tumors occurring in breast tissue are adenocarcinomas where the cancer originates in the luminal mammary epithelial cells that make up the inner lining of ducts and alveoli involved in milk synthesis and transport (Nandi et al., 1995). The most common type of breast carcinoma in women, between 40 to 89 years of age, is invasive ductal carcinoma (81%). The less prevalent type of breast cancer is ductal carcinoma in situ, also known as non-invasive ductal carcinoma (Weaver et al., 2006). The most frequent site of origin of malignant ductal carcinoma is the terminal ductal lobular unit or type 1 lobule, where the unit exhibits high proliferative activity and less differentiation such as branching and clusters (Russo and Russo, 1998). Type 2 and type 3 lobules are more complex in morphology, with more branching and a higher number of ductules per lobule (see Figure 1.1). 1 Figure 1.1 Types of lobule in human mammary gland. A ) Type 1 lobule of nulliparous woman; B) Type 2 lobule of nulliparous woman; C) Type 3 lobule from parous woman. 2 Type 1 and type 2 lobules are structures most frequently found in the breast of nulliparous women of all ages, whereas the more differentiated type 3 lobule predominates in the breast of parous women. After menopause, the mammary parenchyma regresses to structures similar to type 1 lobule (see Figure 1.2). It is postulated that the level of differentiation of lobular structures may be associated with the development of neoplastic lesions with different malignant potential in the breast (Russo and Russo, 2004). 3 Terminal End Buds Approaching Puberty Alveolar Buds , — Type 1 Lobule Puberty Menopause Type 2 Lobule Pregnancy and Lactation After Weaning Type 3 Lobule Type 4 Lobule Figure 1.2 Development of the human mammary gland. Approaching puberty, terminal end buds begin to give rise to alveolar bud and to type 1 lobule. At puberty, type 1 lobule differentiates to type 2 lobule, which comprises of a higher number of ductules per lobule. At the last trimester of pregnancy and lactation period, the mammary gland developed into type 3 and type 4 lobules. After weaning, the structure regresses to type 2 lobule and back to type 1 lobule at menopause. 4 1.2 Etiology and Risk Factors of Human Breast Cancer Breast cancer is a multifactorial disease. There is no single cause of breast cancer. Both endogenous and exogenous factors are involved in breast carcinogenesis. Researchers identified various risk factors associated with breast cancer including age, environment and lifestyle, genetics, and endocrinologic factors. Age is a well-recognized factor for breast cancer development. Breast cancer risk steadily increases with increasing age. The incidence of breast cancer is nearly nonexistent in women younger than 24 years of age and is maximal in post-menopausal women. Environmental and lifestyle factors are also of great importance in the pathogenesis of breast cancer. Unlike cigarette smoking and lung cancer, no causative relationship between environmental or lifestyle factors and breast cancer has been proven, but excessive alcohol consumption, a high-fat diet, increased body mass index in postmenopausal women, as well as excessive radiation exposure are recognized to play a role in the occurrence of breast cancer. Furthermore, familial background or genetics can also influence the risk of developing breast cancer. It is estimated that approximately 5 to 10% of breast cancers are due to a specific inherited mutation in breast cancer susceptibility genes. There are two major hereditary breast-ovarian cancer susceptibility genes, BRCA1 and BRCA2, both of which are tumor suppressor genes (Sakorafas and Pavlakis, 2004, Ganz, 2005). Epidemiological studies strongly support endocrinologic and reproductive factors associated with increased breast cancer risk. These factors include early menstruation, late menopause, nulliparity or late first full-term pregnancy, extended use of oral contraceptives, and prolonged use of hormone replacement therapy (Anderson, 2002, Lewis et al., 2004). A vast majority of women (about 75%), who develop breast cancer have no familial, hereditary, or genetic risk for breast cancer, but these women may have an alteration in tissue response to 5 hormones or a variation in estrogen metabolism (Ganz, 2005). However, the mechanisms whereby estrogens are carcinogenic to the human breast are not completely understood. A better understanding of how estrogens and hormones initiate and regulate breast cancer growth and development is needed to prevent and treat breast cancer. 1.3 Carcinogenicity of Estrogens Epidemiological evidence indicates that early menarche and late menopause are associated with increased breast cancer incidence. Results of the Women Health Initiatives Study (Rossouw et al, 2002) and the Million Women Study (Beral et al, 2003) suggest an overall increase in breast cancer risk from the use of hormone replacement therapy in post-menopausal women. In all of these cases, the level or duration of exposure to estradiol is enhanced. In addition, animal studies provide direct evidence for the role of estrogens in tumorigenesis. Colerangle and Roy (1995) demonstrated that exposure of female Noble rats to diethylstilbestrol and estrone produced a rapid acceleration in cell proliferation and progressively more differentiated epithelial structures in the mammary gland, when compared against control rats. Moreover, prolonged exposure to estrogens was carcinogenic in the liver, kidney, pituitary, and various organs of rats and mice (Weisz et al, 1998, Spady et al, 1999). There are several proposed mechanisms to explain how estrogens are carcinogenic. One mechanism is based on the mitogenic properties of estrogen. Consequently, an increase in cell proliferation enhances the chance for genetic errors during D N A replication, resulting in an increased probability of mutations. Another proposed mechanism suggests that estrogen mediates carcinogenicity through its metabolites, 16a-hydroxy-, 2-hydroxy- and 4-hydroxy-estradiol (Rudali et al, 1975, Liehr et al, 1986, L i and L i , 1987, Lippert et al, 6 2003, Lewis et al, 2005). Accumulating evidence implicates covalent binding of estrogen metabolites to D N A causing mutations leading to cancer (see Figure 1.3, Cavalieri and Rogan, 2004). Furthermore, reactive oxygen species, generated by redox cycling of estrogen metabolites can be genotoxic indirectly (Jefcoate et al, 2000, Yager, 2000). It remains unclear as to which proposed hypothesis contributes to the carcinogenic property of estrogens. 7 OH 4-OH-Estradiol /Estrone o Estrad io l/E stron e-3,4-Qu i no ne DNA Depurinating adducts I Apurinic sites I Mutations I , t . if not \ repaired I Breast cancer Figure 1.3 The bioactivation of estrone and estradiol leading to breast cancer. Estradiol freely converted to estrone in the body by 17p-hydroxysteroid dehydrogenase in the body. Estradiol undergoes bioactivation by CYP enzymes to 4-hydroxyestradiol (4-OH-Estradiol) and Estradiol-3-4-quinones. The reaction of quinone estrogen with D N A to generate the series of events leading to breast cancer. 8 1.4 Hormona l Regulation of Breast Cancer The etiology of breast cancer has a strong hormonal component. Most notably, menstrual status, reproductive history, and estrogen supplementation are risk determinants of breast cancer (Boyle and Leake, 1988). These observations suggest that ovarian and pituitary hormones play an important role not only in the normal breast development, but also in the development of breast cancer and its progression. Treating breast cancer has always been a challenge as there are multiple pathways regulating breast cancer growth. With hormone-dependent breast cancers, hormone deprivation is the principal mean to slow cancer growth. 1.4.1 Ovarian Hormones and Mammary Cancer Estrogen and progesterone are two key hormones produced by the ovary. Estrogen, in particular, is an important hormone in mammary tumorigenesis. The concept of hormone dependency began when physicians observed remission and regression of breast tumors in patients who underwent ovariectomy (Davis, 1958). Later, the discovery of estrogen receptors (ER) in breast tumors confirmed an estrogen signal-transduction pathway in breast tumor growth (Jensen and Jacobson, 1962). These results were further supported by animal studies that showed ovarian hormones stimulated mammary epithelial cell proliferation. Daily 17p-estradiol administration to a particular strain of rat, the ACI rat, caused mammary tumor development (Turan et al, 2004). Dao (1962) demonstrated ovariectomy either prior to or within 7 days of 3-methylcholanthrene (3-MC) or 7,12-dimethylbenz[<3]anthracene (DMBA) administration suppressed rat mammary tumor incidence. A similar observation is seen in humans where 9 the hormonal status of the individual such as pre- or post- menstrual, pregnancy or lactation can significantly affect mammary cancer incidence and multiplicity. Although there is much evidence showing ovarian hormones act as a stimulator or promoter of mammary tumorigenesis, Huggins et al. (1962) demonstrated that the administration of large amounts of 17|3-estradiol (20 Lig) together with progesterone (4 mg) reduced DMBA-induced mammary tumor incidence from 100% in the control group to 52% in the estradiol plus progesterone-treated group. In addition, daily administration of high doses of estradiol, ranging from 20 Lig to 30 mg per rat have been shown to have a protective effect on mammary cancer development and lead to tumor regression in Af-methyl-jV-nitrosourea (MNU)-induced intact rats (Rajkumar et ai, 2001). Cancer can also be controlled by supplying large amounts of estradiol in some breast cancer cases in post-menopausal women (Kennedy, 1962). Estradiol-induced apoptosis was also demonstrated at the cellular level. When MCF-7 cells are grown in estrogen-depleted medium to mimic the hormonal environment of breast tumors of post-menopausal women, treatment with a high concentration of estradiol (> 0.1 nM) promotes a 60% reduction in cell growth (Song et al, 2001). Furthermore, the response of human breast tumors to estrogen changes from stimulatory to inhibitory after prolonged estrogen deprivation according to the new theory of antihormonal resistance evolution (Lewis et al., 2004). Hormone-induced cell apoptosis is a novel concept in breast cancer therapeutic intervention (Lewis et al., 2004). Taken together, these lines of evidence support the dual role of estradiol in mammary cancer growth and development. 1.4.2 Pituitary Factors and Mammary Cancer Ovarian hormones are not the only hormonal factor that regulates mammary tumor 10 growth and development. The use of estrogen deprivation therapies including anti-estrogenic agents such as tamoxifen, aromatase inhibitors, and ovariectomy lead to regression of some but not all breast tumor cases (Lewis et al., 2004). Moreover, patients with advanced breast cancer, who underwent hypophysectomy (surgical removal of the pituitary glands) experienced ovary-independent beneficial effects (Wennbo and Tornell, 2000, Gebre-Medhin et al., 2001). These observations suggest that one or more pituitary factors play a role in breast cancer growth. Animal studies performed by Huggins et al. (1958) also demonstrated a significant regression in tumor size after hypophysectomy in rats with 3-MC-induced mammary cancer, whereas ovariectomy reduced tumor size in most but not all rats. In addition, Sterental et al. (1962) showed that ovariectomy and adrenalectomy resulted in mammary tumor regression in DMBA-induced rats and that administration of estrogen reactivated tumor growth. In contrast, hypophysectomy led to mammary tumor regression but the administration of estrogen failed to reactivate tumor growth. Among pituitary hormones, prolactin (PRL) and growth hormone (GH) are identified as pituitary factors that are primarily responsible for breast cancer growth and development in humans. High circulating levels of PRL and GH have been associated with breast tumor development. It has been shown that human breast cancer and mammary tumor cell lines express growth hormone receptor (Decouvelaere et al., 1995) and prolactin receptor (Murphy et al, 1984) and blocking PRL receptors results in inhibition of growth in cultured mammary tumor cells (Fuh and Wells, 1995). More recently, it was shown that human mammary tumors and normal mammary tissue produce both PRL and G H locally, suggesting a possible autocrine function of the hormones in tumor development and growth. The role of PRL and GH in experimental animal mammary tumors has also been investigated. The importance of GH in mammary tumorigenesis is demonstrated with GH-deficient Spontaneous Dwarf rats. 11 These rats failed to develop mammary tumors when treated with chemical carcinogens such as D M B A and M N U (Swanson and Unterman, 2002). Moreover, exogenous G H or PRL administered to hypophysectomized rats bearing mammary tumors promoted significant tumor growth compared to hypophysectomized rats injected with vehicle (Li and Yang, 1974). However, there are few studies that investigated the effect of individual pituitary hormone in the initiation process of chemical carcinogenesis. Thus, it is undetermined as to whether PRL, GH, or other factors are responsible for the growth of mammary tumors. 1.5 Cytochrome P450 Enzymes Overview The cytochrome P450 (CYP) enzyme system is a superfamily of monooxygenases capable of catalyzing oxidative biotransformation of endogenous compounds and xenobiotics to more water-soluble products. C Y P enzymes are classified into families and subfamilies according to their similarity in amino acid sequence (Nebert et al., 1989). The expression of individual CYP enzyme is tissue and species specific. The CYP enzyme system catalyzes oxidations of a wide range of substrates. These substrates include endogenous compounds such as fatty acids and steroids, and xenobiotics such as drugs and toxins. The biotransformation of these compounds takes place in two phases. During Phase I, enzyme-catalyzed modification adds or exposes a functional group to the parent compound that can be used to attach a conjugate. CYP enzymes are part of the Phase I pathway and catalyze reactions such as hydroxylation, epoxidation, and dealkylation. In CYP-mediated hydroxylation, the added hydroxyl group then serves as the site for further modifications in Phase II metabolism. As mentioned in section 1.3, a proposed mechanism for estrogens to exert its carcinogenicity is via CYP-mediated biotransformation to its carcinogenic estrogen metabolites. Circulating estrogens are metabolized by CYP-mediated hydroxylation (Phase I metabolism) or by direct 12 conjugation to sulfate or glucuronide (Phase II metabolism). A majority of estrogens undergo conjugation, resulting in hormonally inactivated estrogens, whereas a relatively small amount of estrogens are converted to reactive catechol estrogens by C Y P enzymes (Cavalieri etal, 1997). Mammalian species have similar but distinct sets of C Y P enzymes. Between humans, rats, and mice, there are specific C Y P enzymes that share similar function and regulation among species (e.g. CYP1A1, CYP1A2, CYP1B1). However, some C Y P enzymes are unique within a particular species (e.g. CYP2C7, CYP2B2, CYP2C13 in rats). Mammalian CYPs are membrane bound and can be isolated by breaking open the cells and isolating the microsomal membrane (endoplasmic reticulum) fraction. In mammals, the main site of biotransformation takes place in the liver, where C Y P enzymes are the most abundant. Extrahepatic tissues such as lungs, kidneys, brain, and mammary gland also express CYP enzymes, but the expression of CYP enzymes in these tissues is relatively low compared to the liver and not all hepatic forms are found in extra-hepatic tissues. 1.5.1 CYP1 Enzymes Overview In humans and rats, the CYP1 family contains two subfamilies, CYP1A and CYP IB. The CYP1A subfamily contains two members, CYP1 A l and CYP1A2. In rats, constitutive expression of CYP1A enzymes is low in tissues such as small intestine, skin, lung, and liver. In the liver microsomes prepared from uninduced adult male rat, CYP1A enzymes account for approximately 1 to 3% of total C Y P (Ryan and Levin, 1990). More recently, CYP1B1, a single member of the C Y P IB subfamily, had been identified in humans, mice, and rats. CYP1B1 protein is difficult to detect in non-steroidogenic tissues of uninduced animals and its mRNA is present at a very low level in human liver, lymphocytes, endometrium, breast, 13 and lung epithelial cells (Murray, 2001). In the rats, CYP1B1 protein is expressed at a relatively high level in adrenal and testes (Bhattacharyya et al, 1995, Leung et al., 2005). Moreover, tumors of the kidney, prostate, and breast in humans were associated with an increased expression of CYP1B1 proteins (Murray, 2001). The CYP1 enzymes are highly inducible. Induction of CYP1A enzymes is regulated via the aryl hydrocarbon receptor (AhR) (Li et al., 1998). The unliganded AhR exists in the cytosol in a multiprotein complex. Upon binding of the inducing agent (AhR agonist), the multiprotein system dissociates and AhR translocates to the nucleus where it forms a heterodimer with aryl hydrocarbon nucleus translocator. This heterodimer binds to the specific D N A region termed dioxin or xenobiotic response element (DRE or XRE) of the CYP1A gene, thus enhancing its rate of transcription (Vrzal et al, 2004). Chemical inducers of CYP1A enzymes include PAHs (its own substrate), P-naphthoflavone, polychlorinated dibenzo-p-dioxin (PCDD) and other environmental contaminants (Ryan et al, 1982). The magnitude of CYP1A induction can differ depending on the dose of inducer, the structure of the inducer, as well as the target tissue. Humans, rodents, and other vertebrates appear to induce CYP 1A through the same induction mechanism. Therefore, C Y P 1A enzyme induction has become a widely used determinant for exposure to environmental contaminants. The role of CYP1 enzymes in the bioactivation of PAHs, nitrosamines, and aryl amines to their carcinogenic forms has been well documented (Guengerich, 1990). CYP1A induction had been associated with an increased risk in cancer development as an increased level of the enzyme could lead to an enhanced bioactivation of procarcinogens. Similar to the CYP1A enzymes, CYP1B1 is induced by PCDDs and other AhR agonists. Several regulatory elements within the promoter region in the C Y P 1B1 gene have been identified to be structurally similar to the regulatory elements within the CYP1A genes (Murray et al, 14 2001). Although the induction of CYP1B1 expression by AhR agonists has been documented, there are other non-AhR-mediated mechanisms that could also contribute to the regulation of C Y P 1B1. For example, rat CYP 1B1 is inducible by AhR agonists in liver, kidneys, and lungs, and is also inducible by A C T H (adrenocorticotrophic hormone) in the adrenal glands (Bhattacharyya et al., 1995). Although basal expression of CYP1B1 is low-relative to CYP1 A l and CYP1A2 in non-steroidogenic tissues; CYP1B1 protein expression was enhanced in a variety of human cancers including prostate (Tokizane et al., 2005) and colon (Gibson et al., 2003) . Along with emerging evidence supporting the role of CYP1B1 in procarcinogen bioactivation, CYP1B1 expression has become an important determinant of tumorigenesis. 1.6 Epoxide Hydrolase Overview Another Phase I reaction, hydrolysis, is carried out by the enzyme, epoxide hydrolase (EH). Mammalian E H has three forms, cholesterol EH, soluble EH, and microsomal EH. Microsomal E H (mEH) is membrane bound and found in nearly all tissues. However, relative levels vary with tissue, species, sex, and age. Microsomal E H catalyzes the addition of water to epoxides or arene oxides to produce trans-hydroxy(diol) products (Morisseau and Hammock, 2005). The primary role of mEH is to convert foreign metabolically-derived epoxides to diols and in the process converts reactive metabolic intermediates to less mutagenic or carcinogenic products (Hassett et al., 1998). However, mEH also play a role in the activation of PAHs to DNA-damaging diol-epoxides. The inducibility of mEH in mammals is low relative to CYP1 enzymes. Exposure of primary human hepatocytes to compounds such as (3-naphthoflavone and phenobarbital caused moderate increases of less than 3-fold in mEH mRNA expression, whereas CYP1A2 15 and CYP3A mRNA levels were induced more than 10-fold by these compounds (Hassett et al., 1998). In addition, in vivo studies that measured hepatic mEH protein level and enzymatic activity in rats exposed to /rara-stilbene oxide, phenobarbital, Aroclor 1254 or 3-MC also demonstrated modest inductions of mEH (less than 3-fold relative to the control values) (Thomas et al., 1981). Despite the low expression of mEH protein, the presence of mEH is important in the formation of carcinogenic D M B A diol epoxide in the bioactivation of D M B A . 1.7 Animal Model of Human Breast Cancer Animal models are useful experimental systems for the study of mammary cancer. A unique feature of mammary tumors in rats is that the induced tumors are 80 to 90% hormone dependent, whereas spontaneous mammary cancers in dogs and mice are primarily hormone-independent. In humans, approximately one-third of all breast cancer cases are hormone-dependent (Nandi et al., 1995). Mammary cancers can be induced in rats by physical and chemical means including exposing the rats to 1) estrogens, 2) ionizing radiation, and 3) carcinogen chemicals (Huggins, 1965). Chemically induced mammary tumors in rats are, in general, papillary adenocarcinomas (Russo and Russo, 2000). Many are histologically similar to human ductal carcinoma in situ or early invasive adenocarcinomas (Clark, 2002). 1.8 Chemical Carcinogen Induction of Mammary Cancer in Rodents Among different induction methods, the administration of a chemical carcinogen is the most efficient in inducing mammary carcinomas in a large percentage of rats. The most commonly used chemical carcinogens for studying the biology and therapeutic strategies of 16 mammary cancer are M N U and D M B A (Macejova and Brtko, 2001). M N U has been used more recently for the induction of mammary tumors whereas D M B A has been widely used since the 1950's. Critical parameters that affect induction of mammary tumors by chemical carcinogens include the nature and dose of the compound, and the species, strain, age, and hormonal status of the animal (Huggins, 1965). Many research groups have been successful at inducing mammary tumors in virgin adult female Sprague-Dawley rats with a single intragastric administration of 20 mg of D M B A between 50 to 60 days of age. Sprague-Dawley rats are widely used for chemically-induced mammary carcinoma studies because of their high susceptibility to chemical carcinogens. The first palpable mammary tumor usually appears around 8 to 10 weeks post D M B A treatment, and tumor number and size increase with time. Spontaneous mammary neoplastic lesions including fibroadenoma and adenocarcinoma also occur in Sprague-Dawley rats; however, these spontaneous mammary tumors do not appear before 50 weeks of age (Son and Gopinath, 2004). Based on previous experiments, the DMBA-induced Sprague-Dawley rat is a widely accepted animal model with which to study hormonal regulation of human breast cancer. In our laboratory, a single intragastric dose of 20 mg of D M B A had been widely used for the induction of mammary cancer in performing breast cancer studies. By using the same induction method, data such as mammary tumor incidence, multiplicity, latency could be compared between former and future experiments. Therefore, D M B A was chosen to induce mammary tumors in my study. Another chemical carcinogen, M N U , is a direct acting alkylating agent that causes genetic damage or mutations to ras genes, which plays a significant role in signal transduction and regulation of cellular proliferation. The administration of D M B A can be achieved by intragastric and intravenous routes, whereas M N U can be administered by subcutaneous, intravenous, or intraperitoneal injections. 17 (Macejova and Brtko, 2001). 1.9 D M B A Overview Many aromatic organic chemicals are capable of inducing mammary cancer in the rat after a single dose. Aromatic compounds with two or more rings are referred to as polycyclic aromatic hydrocarbons (PAHs). D M B A , which consists of 4 rings, is a type of PAHs (see Figure 1.4) and is relatively potent in inducing skin and mammary tumors in rodents (Cavalieri and Rogan, 2002). Environmental PAHs are generally found in by-products of combustion, in tobacco smoke and in cooked foods. Other examples of PAHs include naphthalene, anthracene, and benzo[a]pyrene (BaP). The carcinogenic activities of PAHs are not associated with the parent hydrocarbon but are a result of the biotransformation of the parent PAH into ultimate carcinogenic metabolites, which covalently bind to D N A causing mutation. Figure 1.4 The chemical structure of D M B A . 18 1.9.1 Bioactivation of DMBA The metabolic activation of D M B A has been studied intensively. There are approximately 30 D M B A metabolites formed by rat liver microsomes (Yang and Dower, 1975). D M B A is a procarcinogen that requires metabolic activation by C Y P and mEH enzymes prior to becoming carcinogenic. CYP and mEH enzymes are located primarily in the liver and are also found in extrahepatic tissues including mammary tissue. Mammary epithelial cells have been shown to be capable of metabolizing D M B A (Christou et al., 1995); therefore, the importance of mammary tissue in D M B A activation should not be neglected. Several investigators observed that the initiation of DMBA-induced mammary carcinogenesis in rodents was altered by pretreating the animals with compounds that affect CYP1 enzyme levels and activities (MacDonald et al, 2001, Chan and Leung, 2003). These studies demonstrated the importance of CYP 1 enzymes in the initiation of mammary carcinogenesis by D M B A . In an in vitro study investigating the effect of different recombinant human and rodent C Y P enzymes in the oxidative metabolism of D M B A , both human and rodent forms of recombinant CYP1 A l were found to be the most active in exhibiting metabolic activity, followed by CYP2C9, CYP2B6, and CYP1A2 (Shou et al, 1996). More recently, several investigators showed that CYP1B1 is required for DMBA-induced cancer using the C Y P l B l - n u l l murine model (Gonzalez, 2001). Furthermore, using human recombinant CYP enzymes, it was demonstrated that CYP1B1 had higher activity than either CYP1 A l or CYP1A2 for the activation of various procarcinogens such as BaP and D M B A (Shimada et al, 2004). Thus, the presence of C Y P enzymes, specifically CYP1, is critical for the bioactivation of D M B A to its carcinogenic metabolites. Previous studies proposed two major pathways of D M B A activation: 1) one-electron 19 oxidation by C Y P or peroxidase enzymes to form intermediate radical cations (RamaKrishna et al, 1992) and 2) metabolic activation to electrophilically-reactive bay-region diol epoxides (Melendez-Colon et al., 2000) (see Figure 1.5). One of these major D M B A activation pathways involves a one-electron oxidation catalyzed by C Y P or peroxidase enzymes to form the reactive carbenium ion at the 12-methyl group. The radical cation intermediate then binds to D N A to form depurinating adduct and ultimately apurinic site. Another major D M B A activation pathway is the bay-region diol-epoxide formation. According to the bay-region theory, formation of the diol-epoxide of D M B A can lead to reactive intermediates that bind to D N A (Shou et al, 1996). DMBA-trans-3,4-diol-l,2- epoxide is formed by sequential reactions in which the first step is CYP-dependent oxidation of the 3,4-position of the D M B A molecule. The oxidation of D M B A at the 3,4-position is mediated most actively by purified C Y P enzymes such as CYP1A1 and CYP1B1 (Shou et al, 1996, Buters et al, 2003). The resulting DMBA-3,4-oxide is then hydrolyzed by mEH to the corresponding trans-dihydrodiol or broken down non-enzymatically to monohydroxyl groups. A second CYP-mediated epoxidation of the 1,2-positions of DMBA-3,4-diol yields the reactive bay region diol epoxide metabolite (see Figure 1.6). 20 Figure 1.5 Metabolic activation of D M B A by one-electron oxidation and diol epoxide pathways. 2 1 Figure 1.6 The bioactivation pathways of DMBA to its carcinogenic metabolite. 1.9.2 Carcinogenic Metabolites of DMBA DNA adduct formation is a critical step in the process by which reactive PAH metabolites cause cancer (see Figure 1.7). Reactive DMBA metabolites covalently bind to DNA to form adducts. The DMBA metabolite-DNA adducts either remain intact in the DNA known as stable DNA adduct or release from DNA by cleavage of the glycoside bond forming depurinating adduct (see Figure 1.8). The unrepaired apurinic site is associated with mutation in genes leading to initiation of cancer. Earlier studies suggested that the 3,4-diol-l,2-epoxide of DMBA is the most active metabolite in binding to DNA in hamster V79 cells (Huberman et al., 1979) and mouse embryo cells (Dipple and Nebzydoski, 1978). However, a limited number of in vivo studies investigated the carcinogenicity of individual DMBA metabolites. An in vivo study involving the topical application of DMBA and its metabolite derivatives on mouse epidermis demonstrated that DMBA-3,4-dihydrodiol derivatives formed the highest number of DNA adducts which further supports the diol epoxide pathway (Schoepe et al, 1986). A more recent study by Melendez-Colon et al. (2000) supports stable DNA-adduct formation by diol epoxides as the primary carcinogenic pathway of PAHs. The study demonstrated that apurinic sites were primarily formed after short periods of exposure of MCF-7 cells to DMBA and BaP. However, when DMBA and BaP were incubated for a longer time (24 hours), stable DNA adducts formed by diol epoxides represented the majority of all DNA lesions, presumably due to an induction of CYP enzyme expression. Most published reports also support the theory that carcinogenic PAHs require CYP and mEH enzyme activity to elicit the formation of mutagenic and carcinogenic diol epoxide metabolites (Chou and Yang, 1978, Huberman et al, 1979) 23 CH 3 D M B A Procarcinogen C Y P I C Y P or Peroxidase Metabolic Activation one-electron oxidation CH 3 3,4-Diol CYP I D N A Adducts Formation | If not repaired Mutation in Genes I Initiation of Cancer Figure 1.7 Overview of-metabolic activation of DMBA leading to cancer 24 H 3 C Stable adduct H,C Depurinating adduct Apurinic site of DNA Figure 1.8 Formation of stable and depurinating DNA adducts and the generation of apurinic sites. 25 1.10 Rationale and Goals of the Present Study The overall goal of the study is to determine the role of hormones in mammary tumorigenesis using the hypophysectomized rat model. A better understanding of the role of hormones in breast cancer growth and development can provide insight into methods of prevention as well as novel treatment therapies. This study was performed to illustrate the importance of hormones in mammary tumorigenesis as well as to validate the use of hypophysectomized rat as in vivo model to study DMBA-induced mammary tumorigenesis. Our study results will provide information on mammary tumorigenesis and expression of DMBA-bioactivating enzymes in intact and hypophysectomized rats. These results will benefit future hormonal studies perform using the hypophysectomized rat model. The goal of this study is: • To determine the effect of pituitary ablation on mammary tumor incidence, tumor burden, tumor multiplicity, and tumor latency. My study will use intact and hypophysectomized female Sprague-Dawley rats as the experimental animal model and DMBA as the chemical carcinogen to induce mammary cancer. Mammary tumorigenesis in the hypophysectomized and intact rats will be monitored and analyzed. 1.11 Experimental Hypotheses 1. Hypophysectomy prevents mammary tumor development in DMBA-treated rats. 2. Expression of CYP and mEH enzymes involved in the bioactivation of DMBA is similar between hypophysectomized and intact rats. 26 12 Specific Objectives To assess mammary tumor incidence, tumor burden, tumor multiplicity, and tumor latency in DMBA-treated hypophysectomized and intact adult female virgin Sprague-Dawley rats To measure CYP1A1, CYP1A2, CYP1B1, and mEH enzyme levels by immunoblot analysis with specific antibodies in liver and mammary tissues of hypophysectomized and intact rats. To determine CYP1-mediated enzyme activities in liver microsomes of hypophysectomized and intact rats using the benzo[a]pyrene hydroxylase assay and methoxyresorufin O-demethylase (MROD) assay. 27 2. M A T E R I A L S A N D M E T H O D S 2.1 Chemicals Chemicals and reagents were obtained from the following sources: Aldrich Chemical Company Inc. (Milwaukee, Wl, USA) Resorufin BD Gentest (Woburn, MA, USA) Anti-rat C Y P 1B1 antibody, cDNA-expressed rat C Y P 1B1 protein BDH Chemicals (Toronto, ON, Canada) Magnesium chloride (MgCl2"6H20) Bio-Rad (Richmond, CA, USA) Bromphenol blue BIOSOURCE International (Camarillo, CA, USA) Alkaline phosphate conjugated, goat F(ab')2 anti-rabbit IgG, gamma and light chain specific, affinity purified Fisher Scientific (Fair Lawn, NJ, USA) Acetone (pesticide grade), acrylamide; ammonium persulphate, N,N'-methylene-bis-acrylamide (BIS), 5-bromo-4-chloro-3-indolyl phosphate p-toluidine salt (BCIP), bovine serum albumin (BSA), dimethylsulfoxide (DMSO), Folin and Ciocalteu phenol reagent, glycerin, glycine, hexane (optima grade), methanol (reagent grade), potassium phosphate (K2HPO4), potassium phosphate monobasic (KH2PO4), sodium 28 carbonate (Na2C03), sodium chloride, sodium dodecyl sulphate (SDS), sodium phosphate (Na2HP04); sodium hydroxide, P-mercaptoethanol, N,N,N',N'-tetramethylehylenediamine (TEMED), 4-nitro-blue tetrazolium chloride (NBT), tris (hydroxymethyl) aminomethane (Tris) J.T. Baker Chemical Co (Phillipsburg, NJ, USA) Sodium dithionite Molecular Probes (Eugene, OR, USA) Methoxyresorufin NCI Chemical Carcinogen Repository Midwest Research Institute 3-hydroxy benzo[a]pyrene Pacific Milk Division (Vancouver, BC, Canada) Skim milk powder Pall Corporation (Pensacola, FL, USA) Nitrocellulose membrane Praxair (Vancouver, BC, Canada) Carbon monoxide gas (99.5% purity) Sigma-Aldrich (St Louis, MO, USA) Benzo[a]pyrene, bromphenol blue; corn oil, cupric sulphate pentahydrate (CuSCvSEbO), 7,12-dimethylbenz[a]anthracene (DMBA), ethylenediaminetetraacetic acid (ETDA), N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES), P-nicotinamide adenine dicucleotide phosphate reduced formed (NADPH), polyoxyethylene sorbitan monolaurate 29 (Tween 20), potassium chloride VWR Scientific Products (West Chester, PA, USA) Blotting paper Dr. S.M. Bandiera (Faculty of Pharmaceutical Sciences, University of British Columbia, Vancouver, Canada) Purified rat cytochrome P450 1A1, purified rat cytochrome P450 1A2, purified rat epoxide hydrolase, rabbit anti-rat cytochrome P450 1A2 serum, rabbit anti-rat epoxide hydrolase IgG 30 Table 2.1 Buffers and reagents used in this study Buffers or reagent Contents Tris-KCl buffer 50 mM Tris; 1.15% KC1; pH 7.4 at 4°C EDTA-KC1 buffer 10 mM EDTA; 1.15% KC1, pH 7.4 at 4°C 0.1 M K P 0 4 buffer (for Total 0.1 M KH2PCVK2HPO4; 20% glycerol; 0.1 m M EDTA, CYP determination) pH7.4 Lowry Reagent C 98% of a; l%ofb ; l % o f c a) 2% N a C 0 3 anhydrous in 0.1 M NaOH b) 2% Na/K tartrate tetrahydrate c) 1 % C U S C V 5 H 2 0 Sample dilution buffer 62.5 mM Tris-HCl, pH 6.8 at room temperature; 10.8% glycerol; 0.001% bromphenol blue; 1% SDS; 5% (3-mercaptoethanol Separating gel 0.375 M Tris-HCl, pH 8.8 at room temperature; 0.1% SDS; 7.5% acrylamide bis; 0.042% ammonium persulphate; 0.03% T E M E D Stacking gel 0125 M Tris-HCl, pH 6.8 at room temperature; 0.1% SDS; 3%> acrylamide bis; 0.08% ammonium persulphate; 0.05% TEMED Electrophoresis buffer (4X) 0.1 M Tris; 0.767 M glycine; 0.4% SDS Transfer buffer (10X) 0.25 M Tris; 1.92 M glycine; 0.1% SDS 10%o Transfer buffer (10X) in 3.5 part distilled water and Transfer buffer (IX) 1 part methanol Modified Phosphate Buffered 1.37 M NaCl; 26 m M KC1, 81 m M Na 2 HP0 4 ; 15 mM Saline (PBS) (1 OX) KH2PO4; 2 mM EDTA Blocking buffer 1% BSA; 3% skim milk powder in modified PBS, pH 7.4 Antibody dilution buffer 1% BSA' 3% skim milk powder; 0.05% Tween 20 in modified PBS, pH 7.4 Wash buffer 0.05% Tween 20 in modified PBS Substrate solution 0.1 M Tris-HCl, 0.5 mM M g C l 2 , pH 9.5 0.1 M HEPES buffer 0.1 M HEPES; 5 mM M g C l 2 , pH 7.8 100 mM KPO4 buffer (for 100 mM KH2PO4/K2HPO4; 5 mM M g C l 2 ; 0.1 mM BaP hydroxylase assay) EDTA, pH 7.5 31 2.2 Animals Forty-nine to fifty-three days old (7 to 7 V2 weeks old) intact and hypophysectomized female Sprague-Dawley rats (140 to 155 g) were purchased from Charles River (Montreal, Canada). Hypophysectomy was performed at 7 weeks of age by the supplier. The animals were acclimatized with monitoring for 3 days post-arrival. Hypophysectomized rats were supplemented with 5% sucrose in their drinking water as a supplement to their diet as recommended by Charles River. Hypophysectomized animals have reduced food consumption when compared against the intact animals; therefore, their drinking water was supplemented with 5% sucrose. The intact animals received regular drinking water. A l l animals in the study had access to food and water ad libitum and were housed in pairs or triplets on corn-cob bedding in polycarbonate cages. Animal quarters were maintained at a temperature of 20 to 23°C and had a 12-hour photoperiod. A l l treatment and experimental procedures were performed in accordance with the principles and policies of the Canadian Council on Animal Care. 32 2.3 Study Designs 2.3.1 Study 1: DMBA-Induced Mammary Tumorigenesis in Sprague-Dawley Rats Study 1 investigated the effect of hypophysectomy on DMBA-induced mammary tumorigenesis. The treatment plan for study 1 is outlined in Figure 2.1. Intact and hypophysectomized female Sprague-Dawley rats were assigned to one of the two treatment groups, corn oil (CO) or D M B A . Each treatment group consisted of 20 animals with the exception of hypophysectomized animals where 27 animals were treated with D M B A . At approximately 60 days of age, intact and hypophysectomized rats were treated with either a single intragastric (i.g.) dose of 20 mg of D M B A (dissolved in 1 ml of corn oil) or an equivalent volume of corn oil vehicle. Body weights were monitored every two days throughout the study period. Mammary tumors development including number, size, and latency was also monitored by palpitation twice weekly starting 5 weeks post treatment. The animals were monitored for 120 to 127 days after treatment and were then terminated by decapitation. Mammary tumors were excised and tumor number, size, location, and weight were recorded. Liver, ovaries, and uterus were also excised and weighed. During the monitoring period, an animal would be scheduled for immediate termination when it 1) developed ulceration or infection of the tumor site, 2) developed tumor mass where it significantly interferes with normal bodily functions or cause pain and distress, or 3) experienced more than 20% loss in body weight of a similar normal animal (taking into account the tumor mass). 33 Hypox (at 49 days of age) Intact At -60 days of age Corn Oil (n = 22) 20 mg DMBA (n = 27) Corn Oil (n = 21) 20 mg DMBA (n = 20) Monitoring for mammary tumors Termination of animals at 120 - 127 days post-treatment Figure 2 .1 Treatment plan for study 1: DMBA-induced mammary tumorigenesis in Sprague-Dawley rats. Hypox: hypophysectomized rats; Intact: Intact rat. 34 2.3.2 Study 2: Expression and Activity of DMBA-Bioactivating Enzymes in Intact and Hypophysectomized Rats Study 2 investigated the effect of hypophysectomy on CYP1 and mEH enzymes expression. Figure 2.3 summarizes the treatment plan for Study 2. Intact and hypophysectomized rats were assigned into one of the two treatment groups, corn oil or D M B A . Each treatment group consisted of 6 animals. At approximately 60 days of age, the animals received either a single intragastric dose of 20 mg D M B A or an equal volume of corn oil (vehicle). Twenty-four hours post dose, the animals were terminated by decapitation and liver and mammary tissue were harvested for microsome preparation and analysis. 35 Hypox (at 49 days of age) Intact At -60 days of age Corn Oil (n = 6) 20 mg DMBA (n = 6) Corn Oil (n = 6) 20 mg DMBA (n = 6) 24 hours post dose, the animals were terminated Liver and mammary tissues were harvested for microsome preparation and analysis Figure 2.2 Treatment plan for study 2: Expression and activity of D M B A - bioactivating enzymes in intact and hypophysectomized rats. 36 2.4 Microsome Preparation 2.4.1 Liver The whole liver was excised and homogenized in cold Tris-KCl buffer in a PotterElvehjem tissue grinder. The homogenate was centrifuged at 9,000 x g for 20 minutes at 4°C to generate the S9 fraction. The S9 fraction was then centrifuged further at 105,000 x g for 60 minutes at 4°C. The supernatant was then discarded and the remaining microsomal pellet was washed and resuspended in EDTA-KC1 buffer using the homogenizer. The resuspended microsomal pellet was centrifuged again at 105,000 x g for 60 minutes at 4°C. After centrifugation, the final microsomal pellet was resuspended and homogenized in a minimal volume of 0.25 M sucrose. A l l samples and test tubes were kept on ice throughout the procedure. The final microsome preparations were stored in small aliquots at -76°C. 2.4.2 Mammary Tissue Mammary tissue was processed similarly to liver as described in section 2.4.1 with the following modifications. The mammary S9 fraction underwent centrifugation at 105,000 x g for 60 minutes at 4°C only once and the pellet was not washed with EDTA-KC1 buffer. The final microsome preparations were stored in small aliquots at -76°C. 37 2.5 Total C Y P Determination Total microsomal C Y P content was determined by the method of Omura and Sato (1964). Microsome samples were diluted in 0.1 M KPO4 buffer, pH 7.4 and divided into two matched spectrophotometer cuvettes (Hellma Canada Ltd). The reducing agent, sodium dithionite (~10 mg), was first added to both cuvettes and then carbon monoxide was gently bubbled into one of the cuvettes. The absorption difference between the two cuvettes was measured at absorption maxima at 450 nm with a SLM-Aminco DW-2C spectrophotometer (Urbana, IL). Total CYP concentration was calculated using the difference between the absorption maximum and the molar extinction coefficient of 91 cm" 1 m M "'. 2.6 Total Protein Determination Total protein content was measured by the method of Lowry et al. (1951). Microsome samples were diluted in water with a dilution factor of 1:20 or 1:10 for liver microsomes and 1:5 for mammary tissue microsomes. Lowry Reagent C was first added to each diluted microsome sample with vigorous mixing. After 10 to 15 minutes, Folin and Ciocalteu's phenol reagent (diluted in 1:1 part with distilled water) was added to each sample. After incubation at room temperature for 30 minutes, absorbance was measured at 750 nm, using a microplate autoreader model EL309 (BIO-TEK Instruments Inc.). Different concentrations (0, 10, 20, 40, and 100 mg per ml) of bovine serum albumin (BSA) were processed by the same procedure and were used to generate a standard curve of absorbance versus protein concentration from which the concentrations of the microsome samples were extrapolated. A l l samples were analyzed in duplicate. 38 2.7 SDS-PAGE and Immunoblot Assay Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) was performed according to the method of Laemmli (1970) using a Hoefer SE 600 vertical slab gel unit (San Francisco, CA). The discontinuous SDS-polyacrylamide gel consisted of a 3% acrylamide stacking gel and a 7.5% acrylamide separating gel. Microsomes were diluted in sample dilution buffer and boiled for 2 minutes. The denatured microsome samples were loaded on to the gel at 20 u.1 per well. Sample loading concentrations are summarized in Table 2.2. Electrophoresis was carried out at a constant current of 23 mA through the stacking gel (approximately an hour) and 46 mA through the separating gel (approximately 2 V2 to 3 hours) until the dye front reached the bottom of the gel. Proteins resolved by SDS-PAGE were transferred electrophoretically onto 0.2 micron pore size nitrocellulose membrane by the methods described by Towbin et al. (1979). The procedure was carried using Hoeffer TE 52 Transphor unit with Power Lid under constant current of 0.4 A for 2 hours in the cold cabinet. After the transfer, the membranes were left overnight in blocking buffer at 4°C. The membranes were incubated with polyclonal rabbit anti-rat CYP1A2 serum, anti-rat CYP1B1 antibody, or anti-rat E H IgG (see Table 2.3 for concentrations used) in antibody dilution buffer at 37°C for 2 hours with shaking, followed by three 5 to 10 minute incubations with wash buffer. The membranes were then incubated with alkaline phosphatase-conjugated goat anti-rabbit IgG (1:3000 dilution) for 2 hours at 37°C with shaking, followed again by three 5 to 10 minute incubations with wash buffer. Colorimetric detection of immunoreactive proteins was completed by reacting with 0.01% NBT and 0.005%) BCIP in substrate solution at room temperature under subdued light. The reaction was stopped by discarding the substrate solution and washing the membrane with distilled 39 water when protein bands were sufficiently stained. The alkaline phosphatase reaction time varied between 1 to 8 minutes. Table 2.2 Amount of microsomal protein loaded per lane for immunoblot assay CYPlAstudy E H study CYP1B1 study Liver Hypox + CO 20 5 — Hypox + D M B A 5 5 ~ Intact + CO 20 5 — Intact + D M B A 5 5 — Mammary tissue f Hypox + CO 40 40 40 Hypox + D M B A 40 40 40 Intact + CO 40 40 40 Intact + D M B A 40 40 40 Values are presented as [xg of total microsomal protein. t Few mammary samples did not have sufficient total protein content to load 40 |ug per lane. For these samples, the maximum amount of total protein was loaded by diluting microsomes with sample dilution buffer in a 1:1 ratio. — indicates immunoblot assay was not performed. Table 2.3 Concentration of antibody used for immunoblot assay Liver Mammary tissue Anti-rat CYP1A2 serum 1:1000 1:500 Anti-rat CYP1B1 ~ 1:500 Anti-rat E H IgG 20 p-g/ml 20 p.g/ml ~ indicates immunoblot assay was not performed. 40 2.8 Quanti tat ion of Immunoblot Staining Staining intensities of protein bands on immunoblots were quantified with a pdi 320 oe scanning densitometer using Quanity One ® version 4.2.0 software (Bio-Rad Laboratories, Hercules, CA). Protein band intensity was measured as contour quantity (CQ), calculated by the software program as optical density x contour area (OD x mm ). The CQ values were divided by the CQ value of a purified standard that was included in each gel. Final protein concentration was calculated using of calibration curve generated by loading various concentrations of purified standards on gels followed by immunoblotting and densitometric analysis as described above. 41 2.9 Enzyme Activity 2.9.1 Methoxyresorufin O-demethylase (MROD) Assay Microsomal methoxyresorufin O-demethylase (MROD) activities were determined by a direct fluorometric method originally described by Burke and Mayer (1974) with modifications. The cytochrome P450-catalyzed demethylation of methoxyresorufin is illustrated in Figure 2.4. Assay mixtures contained 1.93 ml of 0.1 M HEPES buffer, 50 ul of microsomes diluted to 2 mg protein per ml in 0.25 mM sucrose, and 10 \i\ of 0.5 mM methoxyresorufin dissolved in DMSO (2.5 uM final concentration). After a preincubation of 5 minutes at 37°C, the reaction was initiated by the addition of 10 (j.1 of N A D P H dissolved in HEPES buffer (0.25 m M final concentration). The total volume of the reaction mixture was 2 ml and reactions were carried out at 37°C under subdued light. The formation of resorufin was measured as an increase in fluorescence using a Shimadzu RF-540 spectrofluorometer (Kyoto, Japan), with excitation and emission wavelength set at 530 and 582 nm, respectively. The slit width for both the excitation wavelength and emission wavelength were set at 2 nm. Fluorescence values were recorded at 5 minutes post initiation of the reaction. The amount of resorufin formed was determined from a standard curve of fluorescence versus resorufin concentration incubated in the same reaction mixture as the samples with distilled water replacing N A D P H and sucrose replacing microsome. A l l measurements were performed in duplicate. 42 methoxyresorufin resorufin Figure 2.3 The cytochrome P450-catalyzed demethylation of methoxyresorufin 2.9.2 Benzofajpyrene Hydroxylase Assay Benzo[a]pyrene (BaP) hydroxylation activity was measured according to the method described by Nebert and Gelboin (1968). The biotransformation of benzo[a]pyrene to its metabolities is mediated by several CYP enzymes (see Figure 2.5). In this assay, rat hepatic microsomes were incubated with BaP and the formation of BaP metabolites was measured fiuorimetrically. Reaction mixture contained 50 ul of hepatic microsomal protein diluted to 2 mg protein per ml in 0.25 mM sucrose, 500 ul of 100 mM K P 0 4 buffer pH 7.5, 430 ul distilled water, and 10 ul of 8 mM benzo[a]pyrene (80 uM final concentration). After a 5-minute incubation at 37°C, the reaction was initiated by the addition of 10 ul of N A D P H dissolved in distilled water (0.5 m M final concentration). The reaction was carried out in a shaking water bath at 37°C for 4 minutes and stopped with the addition of 1 ml cold acetone to the reaction mixture. Hexane (3.25 ml) was added to each tube for the first extraction. The tubes were capped and mixed vigorously for at least two minutes. The mixture was allowed to settle for 30 minutes prior to transferring 2 ml of the organic fraction to a new test tube. A second extraction with 4 ml of 1 M NaOH was added to the new tubes. The tubes were then shaken vigorously for at least two minutes and centrifuged at 3,000 x g for 10 minutes using a Beckman GP centrifuge (Palo Alto, CA). The upper organic phase was aspirated and 43 discarded. BaP metabolites were measured as the fluorescence of the remaining aqueous phase using a Shimadzu RF-540 spectrofluorophotometer interfaced with a Shimadzu DR-3 data recorder (Kyoto, Japan), with excitation and emission wavelength set at 396 and 522 nm, respectively. The slit width for both the excitation wavelength and emission wavelength were set at 2 nm. The amount of BaP metabolites formed was determined from a standard curve of fluorescence intensity versus concentration generated using known concentrations of a major BaP metabolite, 3-hydroxy-benzo[a]pyrene (3-OHBaP) incubated in the same reaction mixture as the sample with distilled water replacing N A D P H and sucrose replacing microsome. A l l samples were performed in duplicate. benzo [a] py re ne 3-OH-be nzo [a] py rene Figure 2.4 The cytochrome P450-catalyzed hydroxylation of benzo[a]pyrene 2.10 Statistical Analysis The differences in C Y P and mEH enzymes protein levels and enzyme activities were analyzed by two-way A N O V A with Tukey-Kramer multiple comparison test (InStat version 3.00, GraphPad Software, Inc) with the exception in Study 1, where a one-way A N O V A with non-parametric (Dunn) post-hoc test was used to analyze body and organ weights due to non-uniform sample size. Differences with a/?-value < 0.05 were considered to be statistically significant. 44 3. R E S U L T S 3.1 Study 1: D M B A - I n d u c e d M a m m a r y Tumorigenesis in Sprague-Dawley Rats 3.1.1 Effects of Hypophysectomy and DMBA Treatment on Body Weight The animals were weighed every two days throughout the study period. In the beginning of the experiment, intact animals had an average body weight of 170 ± 6 g and hypophysetomized animals had an average body weight of 143 ± 7 g. The average animal body weight of each treatment group is illustrated in Figure 3.1. Hypophysectomized animals had minimal weight gain of less than 33 ± 14 g from beginning to the end of the study. Intact animals, on the other hand, maintained steady weight gain with an average gain of 163 ± 29 g per rat over 120 days. At termination, the body weight of hypophysectomized rats was about 50% of that of the intact animals. There was a significant difference between body weights of intact and hypophysectomized rats. D M B A treatment has a temporary effect on body weight. A single intragastric dose of 20 mg of D M B A or equivalent volume of corn oil was given on Day 0 to both intact and hypophysectomized rats. After treatment, DMBA-treated animals began to lose weight. The total decline in body weight was about 11 ± 7 g (5%>) in the intact animals whereas hypophysectomized rats lost approximately 18 ± 6 g (9.5%>) compared to the weight before treatment. The drop in body weight continued for 4 days after D M B A treatment and body weight gradually returned to the normal pattern. Statistical analysis showed no significant difference between the final body weight of D M B A and corn oil-treated intact rats or hypophysectomized rats (see Table 3.1). 45 400 DMBA Treatment (Day 0) - 0 - Intact + C O (n = 21) H D - Intact + DMBA (n = 20) Hypox + C O (n = 22) - O - Hypox + DMBA (n = 27) I i I I I i t -19 22 42 62 82 Days before and after DMBA treatment 102 122 Figure 3.1 Daily mean body weight of rats in each treatment group (« = 6) during the experimental period of study 1. Values are expressed as mean ± S E M . Table 3.1 Effects of hypophysectomy and DMBA treatment on final mean body weight n Final body weight (g) Intact + CO 21 332 ± 7 Intact + DMBA 20 321 ± 5 Hypox + CO 22 172 ± 3 t Hypox + DMBA 27 169 ± 8 t Values presented as mean ± SEM. Final body weight was measured on the day of termination of rats. f Signficantly different from their corresponding intact groups with a p-value < 0.05. 3.1.2 Effects of Hypophysectomy and DMBA Treatment on Organ Weight At termination, liver, ovaries, and uterus were excised and weighed. The mean organ weights are presented as absolute weight and as a percentage of body weight in Table 3.2. Statistical analysis was performed using the relative (percentage) organ weights to determine the effect of hypophysectomy and DMBA treatment on organ weight. Comparing corn oil-treated intact and hypophysectomized animals, there was no significant difference in liver weight. Conversely, ovary and uterus weights of hypophysectomized animals were significantly lower than those of the intact animals. Liver, ovary and uterus weights of DMBA-treated hypophysectomized animals were not significantly different from their corresponding corn oil-treated groups. According to the results, hypophysectomy affected ovary and uterus weight whereas DMBA treatment had no effect on liver, ovary, or uterus weights. 47 Table 3.2 Effects of hypophysectomy and DMBA treatment on final mean organs weight n Final liver weight (g) Final ovary weight (g) Final uterus weight (g) Intact + CO Intact + DMBA Hypox + CO Hypox + DMBA 21 20 22 27 11.2 ±0.4 (3.36) 11.2 ±0.2 (3.49) 5.5 ±0.1 (3.22) 6.4 ±0.5 (3.77) 0.12 ±0.01 (0.036) 0.16 ±0.02 (0.050) 0.04 ±0.01 (0.023) f 0.03 ±0.01 (0.018) f 0.55 ±0.02 (0.166) 0.52 ±0.03 (0.162) 0.06 ±0.01 (0.035) f 0.06 ± 0.01 (0.035) f Values presented as mean ± SEM. Values in parentheses represent percentage of organ weight per body weight. Final organ weights were measured on the day of termination of rats. Ovary weight was measured from both ovaries. f Signficantly different from Intact+CO and Intact+DMBA groups with a p-value < 0.05. 3.1.3 Effect of a Single Intragastric Dose of 20 mg of DMBA on Mortality A single intragastric dose of 20 mg of DMBA was demonstrated to be very toxic to hypophysectomized rats. Twenty-seven hypophysectomized rats were treated with DMBA and only nine rats survived after the first week (see Table 3.3). Among the intact animals, there were no deaths associated with the same DMBA treatment. A report by Carter et al. (1988) noted DMBA can elicit acute symptoms of lethargy, decreased food consumption, diarrhea, and adrenal necrosis. The deaths seen in the hypophysectomized animals were associated with the acute toxicity of DMBA. Table 3.3 Number of rats in each group before treatment and at termination n Before treatment n At termination Intact + CO 21 21 Intact + DMBA 20 20 Hypox + CO 22 22 Hypox + DMBA 27 9 48 3.1.4 Effect of Hypophysectomy on DMBA-induced Mammary Tumor Incidence Mammary tumor development was monitored for 120 to 127 days (17 to 18 weeks) post treatment. Mammary tumors occurred in 55% of the intact rats treated with DMBA (see Table 3.4). In addition, there was also one case of spontaneous mammary tumor formation in an intact rat in the corn oil group. Mammary tumor latency of the first palpable tumor was approximately 8 weeks after DMBA treatment and tumor number and tumor size (data not shown) increased with time (see Figure 3.2). The number of mammary tumors in the rats was estimated by palpitation and the exact tumor number, tumor size, and tumor location can only be obtained on the day of termination. On average, there were approximately 2.5 mammary tumors per tumor-bearing intact rat. Based on the results of previous studies, mammary tumor incidence will reach 100% in the intact rats treated with DMBA. However, all animals were scheduled for termination on 120 to 127 days post treatment because some tumors were beginning to ulcerate and tumor mass was becoming too large for the animal to bear in accordance to the guidelines recommended by the UBC Animal Care Committee. Table 3.4 Mammary tumor incidence, weight, multiplicity, and latency in intact and hypophysectomized rats following treatment with DMBA and corn oil n Tumor Incidence (%) Average tumor weight (g) Average number of tumor/animal Tumor Latency Intact + CO 21 1(5) 6.18 1 12 Intact + DMBA 20 11 (55) 0.88 ±0.49 2.5 8 Hypox + CO 22 0(0) - - ~ Hypox + DMBA 9 0(0) ~ - ~ Values presented as mean ± SEM (n = 6). Values in parentheses represent percentage of tumor incidence in each group. -- indicates data is not available. 49 o Time (Weeks post treatment) Figure 3.2 Total number of palpable mammary tumors in each group (n = 6) per week post D M B A or corn oil treatment. 3.2 Study 2: Expression and Activity of DMBA-Bioactiviating Enzymes in Intact and Hypophysectomized Rats 3.2.1 Effects of Hypophysectomy and DMBA Treatment on Body and Liver Weight The animals were weighed daily and on the day of termination. Liver weight was collected 24 hours post DMBA treatment. Both intact and hypophysectomized rats were given either a single intragastric dose of 20 mg of DMBA or an equivalent volume of corn oil. Table 3.5 summarizes the effect of hypophysectomy and DMBA treatment on mean body and liver weights. When liver weights were expressed as a percentage of body weight, there was no difference in relative liver weight between the hypophysectomized and intact animals. The results demonstrated hypophysectomy alone did not affect relative liver weight. However, when the animals were treated with DMBA, the relative liver weight of the hypophysectomized animals was lower than that of the intact animals. Our results demonstrated that DMBA treatment along with hypophysectomy significantly reduced relative mean liver weight. Table 3.5 Effects of hypophysectomy and DMBA treatment on body weight and liver weight in female rats terminated 24 hours post treatment. Treatment Body weight (g) Liver weight (g) Liver weight as a percentage of body weight (%) Hypox + CO 172 ± 3 6.41 ±0.26 3.73 ±0.09 Hypox + DMBA 162 ± 1 4.91 ±0.11 3.02 ±0.05 f Intact + CO 212 ± 5 8.14 ± 0.35 3.84 ±0.08 Intact + DMBA 208 ± 4 7.89 ±0.26 3.79 ±0.05 Values presented as mean ± SEM (n = 6). f Significantly different from all other treatment groups with a p-value O.05. 51 3.2.2 Effects of Hypophysectomy and DMBA Treatment on Total CYP Content Protein and total CYP concentrations were measured in hepatic microsomes to determine total CYP content. Total CYP content was expressed as nmol per mg of protein (see Table 3.6). The mean total CYP content was significantly higher in liver microsomes of intact rats treated with DMBA compared with intact rats treated with corn oil. Total CYP content was induced by DMBA treatment in the intact animals. In the hypophysectomized animals, however, there was no difference between corn oil-treated and DMBA-treated groups. The higher CYP content of the corn oil-treated hypo-physectomized rats relative to corn oil-treated intact rats indicates that hypophysectomy increased total CYP content and that DMBA treatment of hypophysectomized rats did not further increase total CYP content. Table 3.6 Effects of hypophysectomy and DMBA treatment on total hepatic CYP content. Treatment Total CYP content (nmol/mg protein) Hypox + CO 0.97 ±0.09 f Hypox + DMBA 1.06 ±0.03 f Intact + CO 0.66 ±0.02 Intact + DMBA 0.98 ±0.05 f Values presented as mean ± SEM (n = 6). f Significantly different from Intact + CO group with a p-value <0.05. 3.2.3 Effects of Hypophysectomy and DMBA Treatment on Hepatic CYP1A2 Protein Expression Hepatic microsomal proteins were resolved by SDS-PAGE. CYP1A1, CYP1A2, CYP1B1 and mEH enzymes in the microsomal samples were detected and quantified by immunoblot analysis as described in section 2.7. The immunoblot was probed with polyclonal rabbit anti-rat CYP1A2 serum, which recognizes both CYP1 A l and CYP1A2 proteins. Figure 3.3 is a scanned image of the immunoblot showing CYP1A1 and 52 CYP1A2 in hepatic microsomes from each treatment group as well as different concentrations of purified CYP1 A l and CYP1A2 standards. CYP1A2 was the lower band (lower molecular weight) and CYP1 A l was the upper band (higher molecular weight). A bar graph summarizing the mean hepatic CYP1A2 protein level for each treatment group is presented in Figure 3.4A. Hepatic CYP1A2 was significantly induced by DMBA in hypophysectomized and intact rats. Compared with the corn oil-treated control group, there was a 8-fold induction of CYP1A2 protein in the hypophysectomized rats and a 7-fold induction in the intact rat samples. The results demonstrated that CYP1A2 was present at similar levels in livers of both intact and hypophysectomized animals and the levels were induced to approximately the same extent by DMBA treatment. 3.2.4 Effects of Hypophysectomy and DMBA Treatment on Hepatic CYP1A1 Protein Expression Hepatic CYP1A1 protein was undetectable in the liver microsomes from corn oil-treated animals whereas hepatic CYP1A1 protein was detected in DMBA-treated animals (Figure 3.3). The results suggested hepatic CYP1 A l protein was induced by DMBA. Protein expression of CYP1 A l in the livers of DMBA-treated intact and hypophysectomized rats was lower than protein expression of C Y P l A2. Figure 3.4B illustrates hepatic CYPl A1 levels for hypophysectomized and intact rats. Statistical analysis showed there was no significant difference between hepatic C Y P l A1 protein levels in the DMBA-treated hypophysectomized and intact rats. 53 (1} CYP1A1 (1)CYP1A1 Standards (2) HYPOX + CO (3) HYPOX + DMBA (4) CYP1A2 standards (5) INTACT + DMBA (6) INTACT + CO standards ( ( ! ( ( ( , , , , ,. . CYP1A1 CYP1A2 Figure 3.3 Immunoblot showing hepatic samples probed with rabbit anti-rat C Y P 1A2 polyclonal serum (1:1000 dilution). CYP1 A l is represented as the upper band and CYP1A2 as the lower band. From left to right, bands showing samples of (1) CYP1 A l standards of 0.05, 0.1, 0.25, and 0.5 pmols per lane, (2) Hypophysectomized rats treated with corn oil, loaded at 2.5 (ag per lane, (3) Hypophysectomized rats treated with D M B A , loaded at 1 u.g per lane, (4) CYP1A2 standards of 0.05, 0.1, 0.25, and 0.5 pmols per lane, (5) Intact rats treated with D M B A , loaded at 1 u.g per lane, (6) Intact rats treated with corn oil, loaded at 2.5 u.g per lane. The membrane was developed with substrate solution for 2 minutes. A. Hepatic CYP1A2 Protein Content 140 B . H C Z o SI z a O -o 2 z o £ 2 O o a. c s f O a 160 140 120 100 80 60 40 4 20 H y p o x - C O Hypox-DMBA Intact-CO Intact-DMBA Hepatic CYP1A1 Protein Content T T Ifllil lIBRs SRIBRI H y p o x - C O Hypox-DMBA Intact-CO Intact - DMBA Figure 3.4 Summary of hepatic CYPl A l (A) and CYP1A2 (B) protein content in corn oil- or DMBA-treated hypophysectomized and intact rats. Bars are shown as mean ± SEM in = 6). * Significantly different from other treatment groups with a p-value <0.05. 55 3.2.5 Effects of Hypophysectomy and DMBA Treatment on Hepatic CYP1B1 Protein Expression CYP1B1 was not examined in the hepatic microsomes because it was demonstrated previously in our laboratory that the protein level was too low to be detected using the immunoblot technique. 3.2.6 Effects of Hypophysectomy and DMBA Treatment on Mammary CYP1A2 Protein Expression Mammary microsome samples were separated by SDS-PAGE and probed with anti-rat CYP1A2 serum to detect CYP1A2 and CYP1A1 enzymes. The amount of microsomal protein loaded per lane for the mammary tissue was 2 to 5 times greater than the liver in order to achieve similar protein band intensity. It suggests that mammary CYP1A2 protein content was much lower when compared with hepatic CYP1A2 protein content in rats. On the immunoblot, CYP1A2 protein bands of corn oil-treated mammary microsome of hypophysectomized and intact animals were poorly visible (see Figure 3.5). Upon treatment with DMBA, CYP1A2 protein was induced in the mammary tissue of both hypophysectomized and intact rats. No statistical analysis was performed because CYP1A2 protein bands were not detected consistently in the mammary microsome samples. Nevertheless, the results demonstrate that mammary tissue expresses CYP1A2 enzyme and that this enzyme was inducible by DMBA in hypophysectomized and intact rats. 3.2.7 Effects of Hypophysectomy and DMBA Treatment on Mammary CYPIA1 Protein Expression On the immunoblot, CYP1 A l protein was undetectable in mammary microsomes from the corn oil-treated animals. Upon treatment with DMBA, mammary CYP1A1 56 protein level was induced in hypophysectomized and intact groups by visual examination (see Figure 3.5). However, mammary CYP1A1 and CYP1A2 protein contents were unable to be determined because the protein bands were not detected consistently in the mammary microsome samples. 3.2.8 Effect of Hypophysectomy and DMBA Treatment on Mammary CYP IB 1 Protein Expression CYP1B1 protein expression was investigated in the mammary microsome samples. Immunoblot analysis showed that CYP1B1 protein was present in the mammary tissue of DMBA-treated hypophysectomized and intact animals, but CYP1B1 protein was not detectable in the corn oil-treated animals. Mammary CYP1B1 protein levels of DMBA-treated intact animals were lower than DMBA-treated hypophysectomized animals according to our immunoblot analysis (see Figure 3.6). Referring to Figure 3.6, protein bands in the "Intact + DMBA" group were visible by eye on the actual blot. However, the band intensities were below the limit of quantitation of the scanner and the image did not appear on the scanned figure. 57 (1) HYPOX + C O (2) HYPOX + D M B A (3) CYP1A2 standards (4) INTACT + D M B A (5) INTACT + C O I 1 I 1 CYP1A1 CYP1A2 Figure 3.5 Immunoblot showing m a m m a r y samples probed w i t h rabbit anti-rat C Y P 1 A 2 po lyc lona l serum (1:500 di lut ion) . C Y P l A l is represented as the upper band and C Y P l A 2 as the lower band. F r o m left to right, bands showing samples o f (1) H y p o p h y s e c t o m i z e d rats treated w i t h corn o i l , (2) Hypophysec tomized rats treated w i t h D M B A , (3) C Y P 1 A 2 standards o f 0.05, 0.1, 0.2, 0.5 p m o l per lane, (4) Intact rats treated w i t h D M B A , (5) Intact rats treated w i t h corn o i l . A l l m i c r o s o m a l samples were loaded at 40 ug o f mic rosomal protein per lane. The membrane was developed w i t h substrate solu t ion for 3.5 minutes. (1) H Y P O X (5) INTACT + C O (2) HYPOX + D M B A (3) CYP1B1 standards (4) INTACT + D M B A + C O I 11 1 I II ' II I CYP1B1 Figure 3.6 Immunoblot showing mammary samples probed with rabbit anti-rat CYP1B1 antibody (1:500 dilution). From left to right, bands showing sample of (1) Hypophysectomized rats treated with corn oil, (2) Hypophysectomized rats treated with D M B A , (3) CYP1B1 standards of 0.05, 0.1, 0.2, and 0.4 pmol per lane, (4) Intact rats treated with D M B A , (5) Intact rats treated with corn oil. A l l microsomal samples were loaded at 40 \xg of microsomal protein per lane. The membrane was developed with substrate solution for 6 minutes. 3.2.9 Effects of Hypophysectomy and DMBA Treatment on Hepatic mEH Protein Expression Microsomal epoxide hydrolase protein level was measured in rat hepatic microsome by probing the immunoblot with rabbit anti-rat EH IgG (Figure 3.7). The antibody detects mEH protein as a single band on the immunoblot. Immunoblot analysis showed that mEH protein was present in the hepatic microsomes prepared from intact and hypophysectomized animals. There was 15 ± 2 u.g of hepatic mEH protein per mg of microsomal protein in control hypophysectomized rats versus 11 ± 3 (.ig of hepatic mEH protein per mg of microsomal protein in control intact rats (Figure 3.9A). Statistical analysis showed mEH protein in the livers of corn oil-treated hypophysectomized rats is significantly higher than that of in the livers of the corn oil-treated intact rats. Comparing hepatic mEH protein expression between corn oil- and DMBA-treated rats, DMBA treatment did not induce mEH in the livers of either intact or hypophysectomized animals. A bar graph in Figure 3.9A summarizes hepatic mEH protein expression in each treatment group. 60 3.2.10 Effects of Hypophysectomy and DMBA Treatment on Mammary mEH Protein Expression I m m u n o b l o t a n a l y s i s s h o w e d t h a t m E H p r o t e i n w a s p r e s e n t i n t h e m a m m a r y t i s s u e o f b o t h i n t a c t a n d h y p o p h y s e c t o m i z e d a n i m a l s . S i m i l a r t o t h e r e s u l t s o f h e p a t i c m E H , m a m m a r y m E H p r o t e i n e x p r e s s i o n is s i g n i f i c a n t l y h i g h e r i n c o n t r o l h y p o p h y s e c t o m i z e d r a t s t h a n i n i n t a c t r a t s . F u t h e r m o r e , D M B A t r e a t m e n t d i d n o t i n d u c e m E H p r o t e i n l e v e l i n t h e m a m m a r y t i s s u e o f e i t h e r i n t a c t o r h y p o p h y s e c t o m i z e d a n i m a l s ( s e e F i g u r e 3 . 8 ) . T h e r e w a s a p p r o x i m a t e l y 0 . 5 ± 0.1 u g o f m E H p r o t e i n p e r m g o f m i c r o s o m a l p r o t e i n i n c o n t r o l h y p o p h y s e c t o m i z e d r a t s v e r s u s 0 .3 ± 0.1 u g o f m E H p r o t e i n p e r m g o f m i c r o s o m a l p r o t e i n i n c o n t r o l i n t a c t r a t s . A b a r g r a p h i n F i g u r e 3 . 9 B i l l u s t r a t e s m a m m a r y m E H p r o t e i n e x p r e s s i o n i n e a c h t r e a t m e n t g r o u p . 61 (1) HYPOX + C O (2) HYPOX + DMBA (3) EH standards (4) INTACT + DMBA (5) INTACT + C O i r Figure 3.7 Immunoblot showing hepatic samples probed with rabbit anti-rat m E H IgG (20 ug/ml). From left to right, bands showing samples of (1) Hypophysectomized rats treated with corn o i l , (2) Hypophysectomized rats treated with D M B A , (3) m E H standards of 0.01, 0.05, 0.1, 0.25, 0.5 ug per lane, (4) Intact rats treated with D M B A , (5) Intact rats treated with corn oi l . A l l microsomal samples were loaded at 5 ug of microsomal protein per lane. The membrane was developed with substrate solution for 2 minutes. (1) HYPOX + CO (2) HYPOX + DMBA ii ir (3) EH standards (4) INTACT + DMBA (5) INTACT + CO Figure 3.8 Immunoblot showing mammary samples probed with rabbit anti-rat m E H IgG (20 p:g/ml). From left to right, bands showing samples of (1) Hypophysectomized rats treated with corn oi l , (2) Hypophysectomized rats treated with D M B A , (3) m E H standards of 0.005, 0.01, 0.05, 0.1, 0.25 u.g per lane, (4) Intact rats treated with D M B A , (5) Intact rats treated with corn oi l . A l l microsomal samples were loaded at 40 p.g of microsomal protein per lane. The membrane was developed with substrate solution for 2.5 minutes. Hepatic mEH Protein Content B. H .E Z 0) m l Z Q-O 15 O 1 z o UJ § 20 '5 15 o CL X 111 E o I E 1 10 * T * T T T i i * * 1 1 liiil^ S 1 I Hypox-CO Hypox-DMBA Intact-CO Intact - DMBA Mammary mEH Protein Content Hypox-CO Hypox-DMBA Intact-CO Intact-DMBA Figure 3.9 Summary of hepatic mEH (A) and mammary mEH (B) protein content in corn oil- or DMBA-treated hypophysectomized and intact rats. Bars are shown as mean ± SEM (n = 6). * Significantly different from other treatment groups with a p-value <0.05. 64 3.2.11 Effects of Hypophysectomy and DMBA Treatment on Hepatic Enzyme Activity Both methoxyresorufin (3-demethylase (MROD) and benzo[a]pyrene (BaP) hydroxylase assays were well established in the laboratory. Assay conditions and equipments had already been optimized and validated in the past by other students and lab members (Ngui, 1997). Additional optimization experiments were performed to substantiate assay conditions for the samples used in the present study. 3.2.11.1 Validation of M R O D assay conditions: Calibration Curve A range of resorufin concentrations, 0, 10, 25, 75, 100, 500, and 1000 pmol per 10 ul, was used to generate the standard curve of fluorescence intensity versus resorufin concentration (see Figure 3.10). A l l fluorescence readings of the sample were within the reading of the lowest and highest resorufin standard concentration. A standard curve was generated each time the assay was performed. 65 Figure 3.10 Calibration curve for the MROD assay. Varying resorufin concentrations of 0, 10, 25, 75, 100, 500, and 1000 pmol per 2 ml of reaction mixture. Assay performed as described in section 2.9.1. A l l measurements were performed in duplicate. 66 3.2.11.2 Validation of M R O D assay conditions: Saturating Substrate Concentration MROD activity was measured in hepatic microsomes of one sample from every treatment group using a range of substrate concentrations, 0, 0.05, 0.1, 0.2, 0.5, 1.0, 1.5, 2 mM. The treatment groups were 1) Intact + CO, 2) Intact + D M B A , 3) Hypox + CO, and 4) Hypox + D M B A . The effect of substrate concentration on resorufin formation at 5 minutes is shown in Figure 3.11. The results suggested that a substrate concentration of 2.5 uM methoxyresorufin (final concentration) was the optimal substrate concentration for all treatment groups. At substrate concentrations above 0.5 uM, resorufin formation decreased. 67 600.0 c 0 2 4 6 8 10 12 Methoxyresorufin concentration (uM) Figure 3.11 Effect of varying substrate concentrations on resorufin formation in hepatic microsomes (2 mg protein per ml) in reaction mixture containing 1.93 ml of 0.1 M HEPES buffer and varying final methoxyresorufin concentrations 0, 0.25, 0.5, 1, 2.5, 5, and 10 uM. Assay performed as described in section 2.9.1. A l l measurements were performed in duplicate. 68 3.2.11.3 Validation of M R O D assay conditions: Reaction Time Varying reaction time for resorufin formation was investigated in rat hepatic microsomes. The MROD activity was measured in a sample prepared from DMBA-treated hypophysectomized rat. Reaction time was investigated under two final substrate concentrations of 1 and 2.5 uM. The results shown in Figure 3.12 suggested that product formation is linear between 0 to 16 minutes. A reaction time of 5 minutes was selected for measuring M R O D activities in the samples. 1400 Time (min) F i g u r e 3 . 1 2 The formation of resorufin over time from 0 to 16 minutes in hepatic microsomes (2 mg protein per ml) in reaction mixture containing 1.93 ml of 0.1 M HEPES buffer and 2.5 uM methoxyresorufin (final concentration). Assay performed as described in section 2.9.1. A l l measurements were performed in duplicate. 69 3.2.11.4 Effects of hypophysectomy and D M B A treatment on M R O D activity In the assay, rat hepatic microsomal samples were incubated with methoxyresorufin under optimal assay conditions and formation of resorufin was measured as described in section 2.9.1. Mean M R O D activities of each treatment group are summarized in Table 3.7. According to the results, hypophysectomy did not affect M R O D activity as there was no difference in M R O D activity in hepatic microsomes prepared from corn oil-treated intact and hypophysectomized rats. On other hand, D M B A induced M R O D activity in hepatic microsomes prepared from both intact and hypophysectomized animals. MROD activity had a 17-fold induction in DMBA-treated hypophysectomized rats compared against uninduced rats and a 14-fold induction in intact rats. The increase in M R O D activity agrees with the induction of hepatic CYP1A2 enzymes by D M B A . A similar result was observed when activities were expressed per nmol of total C Y R The results demonstrated that hypophysectomized rats contain the necessary C Y P enzymes for M R O D activity and the enzymes are responsive to D M B A induction. 70 Table 3.7 Effects of hypophysectomy and D M B A treatment on hepatic microsomal MROD activities Treatment Resorufin formed (pmol/min/mg protein) Resorufin formed (pmol/min/nmol CYP) Hypox + CO 46 ± 6 51 ± 11 Hypox + D M B A 790 ± 58 t 748 ± 62 j Intact + CO 64 ± 1 97 ± 4 Intact + D M B A 896 ± 5 1 t 920 ± 50 § Values are shown in mean ± S E M (n = 6). f Significantly different from Hypox, Intact + CO with a p-value <0.05. I Significantly different from Hypox, Intact + CO, and Intact + D M B A with a p-value <0.05. § Significantly different from Hypox, Intact + CO, and Hypox + D M B A with a p-value <0.05. 3.2.11.5 Validation of BaP Hydroxylase assay condition: Inter-assay variation To eliminate day-to-day variations, a standard curve was generated each time the assay was performed. The product standards underwent the same extraction procedure as the samples to eliminate any discrepancies caused by the extraction methods. 3.2.11.6 Validation of BaP Hydroxylase Assay: Reaction Time Varying reaction time for BaP hydroxylase assay was investigated in rat hepatic microsomes. BaP hydroxylation activity was measured in a sample prepared from corn oil-treated intact rat. Reaction time was investigated from 0 to 10 minutes. The results shown in Figure 3.13 suggested that fluorescence intensity which is a direct measurement of product formation is linear between 0 to 10 minutes. A reaction time of 4 minutes was selected for measuring BaP hydroxylation activities in the samples. 7 1 0.2 Time (min) Figure 3.13 The formation of OH-BaP over time from 0 to 12 mintues in hepatic microsomes (2 mg protein per ml in reaction mixture containing 500 ul of 100 mM KPO4 buffer, 430 ul distilled water and 80 uM benzo[a]pyrene (final concentration). Assay performed as described in section 2.9.2. A l l measurements were performed in duplicate. 72 3.2.11.7 Effects of hypophysectomy and D M B A treatment on BaP Hydroxylation activity Rat hepatic microsomal samples were incubated with BaP under optimal assay conditions and formation of BaP metabolites was measured using Shimadzu spectrofluorophotometer as described in section 2.9.2. BaP is metabolized into many hydroxyl derivatives by C Y P enzymes with the major product as 3-OH BaP. Other minor metabolites are formed at the following relative percentages: 9-OH-BaP, 3-13%, BaP-9,10-diol, 15-25%, BaP-7,8-diol, 12-14%, BaP-4,5-oxide, 8%, and BaP quinones, 14-17%> (Yang 1978). Hepatic BaP hydroxylation activity in rats is mediated by several CYP enzymes. Purified recombinant human CYP1A1 and CYP1B1 exhibit the highest BaP hydroxylation activities versus recombinant CYP2A6, CYP2C9, CYP2C19, and CYP3A4 (Shimada 2004). Mean BaP hydroxylation activities of hepatic microsomal samples in each treatment group are summarized in Table 3.8. BaP hydroxylation activity is expressed as both nmol of OH-BaP formed per min per mg of protein and also per nmol of total CYP. Between the BaP hydroxylation activity of the control animals, no difference is observed in either intact or hypophysectomized rats. D M B A treatment induced BaP hydroxylation activity in intact and hypophysectomized rats. BaP hydroxylase activity (expressed as per mg of protein) had a 3-fold induction in the DMBA-treated hypophysectomized rats, whereas BaP hydroxylase activity had a 5-fold induction in the DMBA-treated intact rats. The magnitude of induction in BaP hydroxylase activity is significantly different in the liver microsomes prepared from DMBA-treated intact and hypophysectomized animals. Although the activities were different between DMBA-treated hypophysectomized and intact rats, the results suggested that hypophysectomized rats contain the C Y P enzymes needed for the hydroxylation of BaP and D M B A was able to induce these C Y P enzymes in the livers of 73 intact and hypophysectomized rats. Table 3.8 Effects of hypophysectomy and D M B A treatment on hepatic microsomal BaP hydroxylase activities. Treatment OH-BaP formed (pmol/min/mg protein) OH BaP formed (pmol/min/nmol CYP) Hypox + CO , Hypox + D M B A Intact + CO Intact + D M B A 65 ± 10 180 ± 20 t 62 ± 10 330 ± 3 0 % 190 ± 2 0 4 3 0 ± 5 0 | 230 ± 2 0 850 ± 8 0 $ Values are shown in mean ± S E M (n = 6). f Significantly different from Hypox, Intact + CO, Intact + D M B A with a p-value O.05. % Significantly different from Hypox, Intact + CO, Hypox + D M B A with a p-value <0.05. 74 4. DISCUSSION Rats are a widely used animal model for studying human breast cancer as the histology of mammary tumors in rats closely resembles human breast cancer (Russo et al., 1990). A single treatment with D M B A has been reported to be highly effective at inducing mammary cancer in intact virgin rats. However, D M B A is not as effective in inducing cancer in hormonally-depleted (i.e. ovariectomized or hypophysectomized) rats (Huggins et al., 1958). The present study investigated the effect of hypophysectomy and D M B A treatment on mammary tumorigenesis and on the expression of hepatic and mammary C Y P l and mEH enzymes in female Sprague-Dawley rats. Previous studies reported that hypophysectomized rats failed to develop mammary cancer following treatment with 3-MC. Both 3-MC and D M B A require metabolic activation to become carcinogenic. Specifically, D M B A requires C Y P l and mEH enzymes to form the carcinogenic metabolite, DMBA-3,4-diol epoxide. Our hypothesis was that hypophysectomy would reduce mammary tumor incidence in DMBA-induced rats. Hypophysectomized rats may lack specific CYP enzymes required for D M B A bioactivation. For the second hypothesis, our study aimed to test CYP enzyme expression and activity for the bioactivation pathway of D M B A between hypophysectomized and intact rats. Two experiments were performed. Study 1 investigated the effect of a single D M B A treatment on mammary tumorigenesis in intact and hypophysectomized rats. Study 2 analyzed the expression of hepatic and mammary CYP and mEH enzymes in hypophysectomized rats. In addition, a separate study examined body weight and mortality in hypophysectomized rats given intermittent D M B A treatment (see Appendix I). 75 4.1 Effect of Hypophysectomy on Growth and Organ Development Body weight of the animals was measured throughout the study as an indicator of the animals' health. Hypophysectomized animals exhibited little or no weight gain when compared to intact animals. Hypophysectomized rats exhibited a mean weight gain of 33 ± 14 g over 120 days, whereas intact rats had an average weight gain of 163 ± 29 g. Growth hormone is one of the pituitary hormones that stimulates endogenous production of IGF-1 (insulin-like growth factor-1) leading to somatic growth stimulation. Body and some organ weights are under the influence of growth hormone and IGF-1 (Guler et al., 1988). Monitoring the body weights of intact and hypophysectomized animals also provides an indication of the success of the surgical procedure of hypophysectomy. Hypophysectomy affects organ development differentially. Guler et al. (1988) reported that kidney and spleen weights were reduced in hypophysectomized rats and that administration of IGF-1 to these rats restored organ weights to their normal range. In our study, liver, ovary and uterus were excised and weighed at the time the rats were terminated. There was no difference in relative liver weight but relative ovarian and uterine weights were significantly lower in hypophysectomized rats when compared to intact rats. Development of the ovaries and uterus is under the influence of L H and FSH, which are secreted by the pituitary gland. The administration of recombinant human FSH to hypophysectomized animals was shown to restore ovarian and uterine weights to values that are similar to organ weights in intact animals (Mannaerts et al., 1994). Furthermore, FSH also induced follicular growth to preovulatory stages, induced ovarian estradiol production, and induced endometrial proliferation in hypophysectomized rats. Mammary gland development is also under the influence of pituitary hormones. In rats, 76 the mammary glands are distributed in pairs along the milk line, with one pair located in the cervical, two pairs in the thoracic, one pair in the abdominal and two pairs in the inguinal regions (Russo et al., 1990). Mammary gland development is a progressive process which begins during the neonatal period. The mammary gland, before puberty, is characterized by branches of ducts with terminal end buds (TEBs). At puberty (between days 35 and 42 of age), which is defined by the onset of the estrous cycle, TEBs in the mammary gland begin to differentiate to alveolar buds and lobules and lobule formation accumulates over multiple estrous cycles (Masso-Welch et al., 2000). Similar to the development of the human breast, the lobules in virgin female rats are predominantly type 1 lobules with little branching and differentiation. During pregnancy and lactation, the lobules increase significantly in size and in the number of alveoli to a stage called type 3 and type 4 lobules (Russo et al., 1990). Experimental evidence shows the importance of ovarian and pituitary hormones in mammary gland development (Reece et al., 1936, Nathanson et al., 1939). The proliferation of mammary epithelium is induced by ovarian hormones, estradiol and progesterone. However, mammary gland proliferation can only respond to ovarian hormones i f the pituitary gland is intact. The mammary gland shows almost complete atrophy in rats following hypophysectomy (Nathanson et al., 1939). Ovarian hormones alone have little or no mammogenic activity (Lamote et al., 2004). Pituitary hormones that have been shown to be involved in the stimulation of mammary duct growth include growth hormone, prolactin, and adrenal corticoids (Nandi et al., 1995, Lamote et al, 2004), but the underlying mechanism for each hormone remains unsolved. A study using hypophysectomized monkeys further demonstrated the complex role of the pituitary gland in mammary mitogenesis (Kleinberg et al., 1985). In that study, it was demonstrated that hypophysectomy prevented full estradiol-induced mammary development. Mammary gland development was significantly 77 decreased in estradiol-treated hypophysectomized monkeys compared to estradiol-treated intact monkeys. Thus, it is evident that the pituitary plays an important role in mammary gland development in rodents and primates. 4.2 Effect of D M B A Treatment on M a m m a r y Tumorigenesis A single intragastric dose of 20 mg of D M B A is conventionally used to induce mammary cancer in Sprague-Dawley rats. The type of mammary tumor induced by D M B A in rats is primarily papillary carcinoma, a type of malignant lesion that originates in the mammary ducts (Russo and Russo, 2000). D M B A induces tumors with latencies that range between 8 and 21 weeks. Final tumor incidence is close to 100% if the animals were monitored for 180 days post-treatment (Russo and Russo, 1996). The susceptibility of the mammary gland to DMBA-induced carcinogenesis is strongly age-dependent. Susceptibility is maximal when the carcinogen is administered to animals between the ages of 45 to 55 days, the age of sexual maturity. A decline in tumor incidence and number of tumors per animal is observed when the animal is treated after this period (Sinha et al., 1983). In our study, rats were treated with D M B A at approximately 60 days of age. The animals were monitored for mammary tumors for 120 to 127 days following D M B A treatment before the termination of the animals. The study period was not extended beyond 127 days because mammary tumors in some of the rats grew to a size that affected the animal's mobility. In addition, a few tumors were starting to ulcerate through the adjacent skin at 127 days after treatment. Thus, in compliance with guidelines of the university animal care committee, we decided to terminate all rats at 120 to 127 days after D M B A treatment. At 120 to 127 days post D M B A treatment, intact rats had a 55% tumor incidence with the first palpable tumor at 8 weeks post D M B A treatment. Tumor incidence (55%) was less than the 80 to 100% value reported in the literature. This could be 78 as a result of the following reasons: 1) the study period for my study was only 120 to 127 days, whereas previous studies monitored the animals for 180 days or longer and 2) the rats in my study were dosed at approximately 60 days of age; however, rats are the most susceptible to carcinogenic chemicals between 45 to 55 days of age. In terms of tumor multiplicity, there was an average of 2.5 tumors per tumor-bearing rat. DMBA-induced mammary incidence, latency, and multiplicity were consistent with previous data obtained in our laboratory (Tai et al, 2006). 4.3 Effects of Hypophysectomy and D M B A Treatment on Mammary Tumorigenesis We initially hypothesized that hypophysectomized rats treated with D M B A would develop fewer mammary tumors than intact rats. A single intragastric dose of 20 mg of D M B A was given to both intact and hypophysectomized adult female virgin Sprague-Dawley rats. None of the DMBA-treated hypophysectomized rats developed mammary tumors during the monitoring period, whereas DMBA-treated intact animals had a 55% mammary tumor incidence. Our results highlight the importance of the pituitary in the initiation and development of mammary tumors in DMBA-treated rats. A previous study demonstrated that treatment with 3-MC induced mammary cancer in 67% of ovariectomized rats versus 100%o in intact rats, whereas none of the 3-MC-treated hypophysectomized rats developed mammary tumors (Huggins et al, 1959). Furthermore, Sterental et al. (1962) determined that both ovariectomy and hypophysectomy caused mammary cancer regression; however, estrogen administration failed to reactivate tumor growth in hypophysectomized rats whereas estrogen administration reactivated tumor growth in ovariectomized rats. Our study together with previous studies demonstrates that both ovarian and pituitary hormones are involved in 79 the regulation of breast cancer, but an intact pituitary appears to be an essential component in mammary tumor growth and development. Further experiments are needed to identify the individual or a combination of pituitary factors that are involved in mammary tumorigenesis. 4.4 Adverse Effects Associated with D M B A Treatment In the present study, we assessed the effect of D M B A treatment on body weight and mortality. D M B A treatment had a short-term effect on body weight. Both intact and hypophysectomized animals experienced a decline in body weight for 4 days after the administration of a single intragastric dose of 20 mg of D M B A . In addition, this D M B A dose caused the deaths of 18 DMBA-treated hypophysectomized animals within the first week of treatment. Acute toxic effects of D M B A include lethargy, diarrhea, decreased food consumption, and adrenal necrosis that develop within a day after D M B A treatment (Carter et al, 1988). Previous studies reported that administration of D M B A at a dosage of 27 mg per 100 g of body weight caused death in one-half of the treated rats (Huggins et al., 1961). At this dose, adrenal necrosis was found 3 days after intragastric administration of D M B A and adrenal regeneration began 6 or 7 days post-DMBA treatment (Huggins etal, 1961). The researchers involved in that study concluded both intravenous and intragastric administrations of D M B A cause adrenal necrosis, diarrhea, as well as weight loss in rats. Although weight loss was seen in both intact and hypophysectomized rats treated with 20 mg of D M B A in our study, the same D M B A treatment only caused deaths in hypophysectomized rats. We conclude that hypophysectomized animals are more susceptible to the toxic effects of 20 mg of D M B A because D M B A treatment produced adrenal necrosis, diarrhea, and reduced food consumption (i.e. physiological stresses) that these rats could not overcome because they 80 lacked the pituitary hormones needed to restore homeostasis. A different D M B A dosing approach that would be tolerable while preserving mammary carcinogenicity was investigated in the hypophysectomized rats (see Appendix I). The 20 mg dosage of D M B A was divided into four 5 mg doses given intermittently to hypophysectomized rats over a 2-week period. Previous studies demonstrated that divided D M B A dosing was as effective as a single 20 mg dose of D M B A for inducing mammary cancer in rats (Hollingsworth et al., 1998). Hollingsworth et al. (1998) administered 5 mg of D M B A (dissolved in 1 ml of corn oil) weekly for a total dosage of 20 mg D M B A to intact Sprague-Dawley female rats {n = 9). In that study, 7 of 9 rats (77.8%) developed malignant tumors, with an average of 2.3 tumors per affected rat. Using the intermittent dosing approach, no significant change in body weight was observed between each D M B A dose and no death was reported in the treated hypophysectomized rats in our study. The acute toxicity of D M B A appears to be dose dependent. Our results demonstrated that hypophysectomized rats can tolerate D M B A administered as four doses of 5 mg each. Although hypophysectomized rats tolerated the divided D M B A dosing regimen, the animals were more difficult to handle on the days they were to receive the third and fourth intragastric doses. The animals had experienced unpleasant adverse effects after the first and second doses of D M B A and they began to reject upcoming doses. For future experiments, the total 20 mg dosage of D M B A could be divided into two doses of 10 mg each given once a week to hypophysectomized rats to induce mammary tumors. 81 4 . 5 Bioactivation of D M B A in Hypophysectomized Rats Both D M B A and 3-MC require metabolic activation by C Y P and mEH enzymes to form carcinogenic metabolites (Christou et al., 1989, Shou and Yang, 1990, Shimada and Fujii-Kuriyama, 2004). Failure of the hypophysectomized rats to develop mammary tumors could be as a result of down-regulation of specific CYP and mEH enzymes, which are needed to metabolically activate D M B A . Hepatic metabolism in rats is known to be influenced by gonadal and pituitary hormones. Specifically, expression of hepatic CYP2A, CYP2C, CYP2B, CYP3A, and CYP2E1 enzymes is altered in hypophysectomized rats (Oinonen et al, 1995, Agrawal and Shapiro, 1997, Chen et ai, 1999). Expression of extrahepatic CYP enzymes is also regulated by gonadal and pituitary hormones. In our laboratory, we demonstrated that L H and FSH play a role in the regulation of testicular C Y P IB 1 expression (Leung et al, 2005). Hence, the loss of pituitary hormones in the hypophysectomized animals could lead to an alteration of hepatic and mammary expression of C Y P and mEH enzymes required for D M B A activation and ultimately to an inability to develop mammary tumors. To the best of my knowledge, there are no previous studies that examined the biotransformation of D M B A in hypophysectomized female rats. A previous report compared hepatic metabolism of D M B A between male, female, and ovariectomized Sprague-Dawley rats (Vater et al., 1991). D M B A was infused into the liver and the production of the carcinogenic metabolite, DMBA-3,4-dihydrodiol, was measured in the perfusate. DMBA-3,4-dihydrodiol appeared in the perfusate at higher rates in intact and ovariectomized female rats than in the male rats. The results suggested that the hepatic enzyme composition was different between male and female rats and a possible explanation for a lower incidence 82 of DMBA-induced mammary tumors observed in male as opposed to female rats (Dao, 1962). In the present study, we measured the expression and activity of C Y P and mEH enzymes, which have been shown to be involved in the bioactivation of D M B A (Christou et al, 1995, Shimada and Fujii-Kuriyama, 2004), in intact and hypophysectomized female rats. 4.6 Effect of Hypophysectomy on C Y P 1 and m E H Enzyme Expression CYP1 and mEH enzyme expression was measured in hepatic and mammary microsomes prepared from Sprague-Dawley female rats treated with a single intragastric dose of 20 mg of D M B A or an equivalent volume of corn oil. Rats were terminated 24 hours post treatment. Enzyme induction by PAHs such as 3-MC was demonstrated to be maximal at 24 hours post treatment (Conney, 1967). Liver and mammary gland were excised for microsome preparation. The CYP 1A family enzymes are well documented to be involved in the bioactivation of D M B A (Guengerich, 1990). Moreover, formation of the potent carcinogenic D M B A diol-epoxide metabolite requires the presence of mEH. The main site for D M B A biotransformation is the liver, which is involved when the compound goes through first pass metabolism. Liver contains the majority of enzymes required for the biotransformation of xenobiotics and endogenous compounds. There is evidence that hepatic metabolic activation of nitrosamine or PAHs produce short-lived electrophiles that could damage D N A in extrahepatic tissue (Williams and Phillips, 2000). Therefore, the activities and level of CYP1A and other enzymes in the liver were examined. In rats, D M B A induces tumor formation primarily in the mammary gland. Recent research suggested that bioactivation of mammary carcinogens proceeds through a primary metabolic step carried out by hepatic metabolism, followed by complete metabolic activation to the ultimate DNA-reactive 83 metabolite in the breast (Williams and Phillips, 2000). Although the relative contribution of hepatic and mammary carcinogen activation have not been determined, levels of enzymes in the mammary glands are also important in influencing the carcinogenicity of D M B A . Hepatic CYP1A1 and CYP1A2 protein expression was measured in liver microsomes prepared from intact and hypophysectomized rats. The CYP1A1 protein band was undetectable in liver microsomes from corn oil-treated intact and hypophysectomized rats, whereas the C Y P l A2 protein band was easily detected. The results demonstrated that basal expression of hepatic CYP1A1 was lower than basal expression of hepatic CYP1A2. Our study results agree with previous data where the expression of C Y P l A2 and CYP1A1 protein in control Sprague-Dawley female rat liver is 31 ± 9 and 9 ± 4 pmol per mg microsomal protein, respectively (Walker et al., 1999). No difference in hepatic CYP1A2 expression was found between hypophysectomized and intact rats. Expression of hepatic CYP1B1 protein was not examined in this study because hepatic CYP1B1 protein levels in uninduced rats have been shown to be below the limit of detection (Walker et al., 1999). CYP1B1 protein has only been detected in liver of rats given chronic 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) treatment at doses more than 35.7 ng/kg/day for 30 weeks (Walker et al, 1999). Basal mammary CYP1A1, CYP1A2, and CYP1B1 protein expression was also examined in corn oil-treated rats using the immunoblot technique. However, mammary CYP1A1, CYP1A2, and CYP1B1 protein was detected inconsistently on the immunoblots. By approximation of the amount of microsomal protein sample loaded in the immunoblot, mammary levels of CYP1A1 and C Y P l A2 enzymes were more than 2 to 5 times lower than in the liver. C Y P l A , 2 A , 2B, 2E1, 3 A, 4A proteins or mRNA were also found in the breast of human and rat, but the level of expression of these enzymes was lower than in the liver 84 (Hellmold et al, 1995). As demonstrated by our results and other studies, mammary CYP 1A1, 1A2, and CYP1B1 protein expression was often too low to be detected using immunoblot analysis (Christou et al, 1995). Other studies examined the expression of mammary CYP1A and CYP1B1 enzymes in rat mammary tissue and human breast epithelial and tumor cells by measuring mRNA levels through reverse transcription-PCR (RT-PCR) (Spink et al, 1998, Badawi et al, 2000). In terms of mRNA expression levels, mammary CYP1A2 had the highest expression followed by CYP1 A l and CYP1B1 in the control rat. The level of CYP1A2 mRNA in the mammary gland of the control rat is 43-fold higher than CYP1A1 and 13000-fold higher than CYP1B1. Measuring C Y P enzymes by mRNAlevel could provide information on the relative expression of individual C Y P enzyme in tissue. However, the level of mRNA expression does not always reflect the level of protein expression (Iba et al, 1999). Our results indicate that hypophysectomy does not affect CYP1 enzyme expression in hepatic and mammary tissues. Basal mEH protein levels were measured in liver and mammary microsomes of hypophysectomized and intact female rats. Hepatic mEH protein expression was approximately 1.3-fold higher in corn oil-treated hypophysectomized rats than control intact rats. Mammary mEH protein levels were approximately 1.7-fold higher in the corn oil-treated hypophysectomized rats than corn oil-treated intact rats. Comparing mEH expression level in liver and mammary tissue, mEH protein expression is more than 30 times higher in liver than in the mammary gland. Our study was the first to measure the expression of mEH protein in mammary tissue of hypophysectomized female rats. The results demonstrated an up-regulation of mEH enzymes in liver and mammary tissue by hypophysectomy suggesting that the regulation of mEH is influenced by pituitary hormones as proposed by other researchers (Delinger and Vesell, 1989, Inoue et al, 1995). 85 4.7 Effect of DMBA Treatment on C Y P 1 and mEH Enzyme Expression Both CYP1A1 and CYP1A2 protein levels were induced in the livers of hypophysectomized and intact rats after a single intragastric dose of 20 mg of D M B A . Our results were consistent with other studies, which demonstrated that PAHs and TCDD are capable of inducing CYP1 enzymes through the Ah receptor (Walker et al, 1999, Badawi et al, 2000). CYP1A2 and CYP1A1 enzymes expressed in hepatic tissue were induced by D M B A treatment as shown by immunoblot analysis. The immunoblot data also showed that basal CYP1A2 and CYP1 A l protein levels were similar in the livers of intact and hypophysectomized rats. D M B A appears to induce CYP1 A l to a greater extent than CYP1A2 in the liver. Badawi et al. (2000) measured CYP1 enzyme mRNA levels in untreated and chlorinated hydrocarbon-treated rat livers. In that study, mRNA levels of CYP1A1, CYP1A2, and CYP1B1 were induced in the liver of rats administered TCDD, dieldrin, or 2,4-dichlorophenoxyacetic acid. There are no published studies that investigated the induction of hepatic or mammary CYP1 enzymes by a single dose intragastric dose of 20 mg of D M B A . In a previous study, D M B A was administered to mice as a single intraperitoneal injection at dosages of 5 to 100 mg per kg of body weight and induction of hepatic mRNA levels of CYP1A1, CYP1A2, and CYP1B1 was observed. The study also stated that D M B A , in comparison with other PAHs such as 3-MC, benzo[ft]fluoranthrene, and BaP, is only a moderate inducer of CYP1 A l and CYP1B1 in mouse liver (Shimada et al, 2003). In mammary tissue, CYP1A1, CYP1A2, and CYP1B1 enzyme expression was induced by D M B A treatment as shown by immunoblot analysis. We were unable to quantify the basal expression of mammary CYP1 protein because band intensity was below the limit of 86 quantitation. By visual inspection, the expression of mammary CYP1A2, CYP1A1, and CYP1B1 were induced by D M B A treatment in hypophysectomized and intact rat. The level of mammary CYP1A2 is the highest, followed by CYP1A1 and CYP1B1. Our study demonstrated that hypophysectomized animals are capable of inducing CYP1A1, CYP1A2, and CYP1B1 expression in liver and mammary tissue. Microsomal E H protein levels were not induced by D M B A treatment in liver and mammary tissue of hypophysectomized and intact female Sprague-Dawley rats. A previous study reported a modest induction of mEH in the livers of rats administered ^rara-stilbene oxide (a synthetic proestrogen), phenobarbital, Aroclor 1254, or 3-MC (Thomas et al, 1981). However, our results showed mEH expression was unaffected by D M B A treatment in liver or mammary tissue. In the study by Thomas et al. (1981), rats were treated with daily intraperitoneal injections of the chemical for 4 days and the animals in our study were treated with a single intragastric dose of D M B A . Thus, it is possible that the dose of D M B A and the duration of treatment in our study were insufficient to elicit induction of mEH. 4.8 Methods to Measure D M B A Bioactivation In the present study, we did not directly measure D M B A bioactivation in intact and hypophysectomized female Sprague-Dawley rats. It would be ideal to measure D M B A biotransformation directly in the liver and mammary tissue to identify possible differences between hypophysectomized and intact rats. There are different approaches to determine the bioactivation of D M B A in vivo and in vitro. Previous studies incubated D M B A with rat liver or mammary microsomes in a reaction mixture and oxidative metabolites of D M B A were measured and identified using HPLC (high pressure liquid chromatography) (Tamulski et al, 87 1973, Yang et al, 1975, Wong et al, 1980). Vater et al (1991) analyzed the formation of D M B A metabolites using non-recirculating liver perfusion, where the liver was left in situ and the common bile duct, portal vein, and vena cava were cannulated for perfusion. D M B A in the perfusion medium was then infused into the liver and the perfusate was collected for HPLC analysis of D M B A metabolite formation. Alternatively, formation of D M B A - D N A adducts in the collected sample could be analyzed using HPLC as previously described by Cai et al (1997) and Kleiner et al (2002). The D M B A metabolites can be resolved by H P L C and can be detected by U V or fluorescence spectroscopy and identified by comparison of their retention times with those of synthetic reference compounds. D M B A metabolites were identified by comparing the chromatographic properties of the reference compounds. Some, but not all, synthetic D M B A metabolites are available commercially. At this time, a D M B A biotransformation assay has not been established in this laboratory. For the present study, we analyzed the potential for D M B A bioactivation in hypophysectomized and intact rats indirectly by measuring the activities of hepatic C Y P l enzymes that are documented to be involved in the bioactivation of D M B A . 4.9 Effects of Hypophysectomy and D M B A Treatment on Enzyme Activities MROD and BaP hydroxylase assays were used as a measure of CYPlA-mediated enzyme activities in hepatic microsomes. MROD and BaP hydroxylase activities were not measured in mammary microsomes due to inadequate sample size for analysis. Approximately 150 ul of microsome was prepared from mammary tissues per rat. The protein concentrations in the mammary samples were between 5 to 10 mg of microsomal 88 protein per ml. However, each immunoblot or enzyme activity assay required 20 to 40 ul of microsomal sample. In our study, we decided to use the mammary microsome samples for immunoblot analysis to determine C Y P l and mEH protein levels. MROD and ethoxyresorufin 0-deefhoxylase (EROD) assays have been used as an indicator of C Y P l A2 and CYP1A1 activity, respectively. Burke et al. (1994) determined that the demethylation of methoxyresorufin is catalyzed primarily by C Y P l A2 in liver microsomes prepared from control rats and 3-MC-treated rats. Ethoxyresorufin was a selective probe for C Y P l A1 in liver microsomes prepared from 3-MC-treated rats. Benzo[a]pyrene is a PAH that is often used as a substrate for analyzing microsomal C Y P l A. BaP is oxidized into many hydroxyl derivatives by C Y P enzymes with 3-OH BaP as the major product. Minor metabolites are formed in the following relative percentages: 9-OH-BaP, 3-13%, BaP-9,10-diol, 15-25%, BaP-7,8-diol, 12-14%, BaP-4,5-oxide, 8%, and BaPquinones, 14-17% (Yang and Kicha, 1978). Analogous to D M B A , BaP requires C Y P and mEH enzymes for activitation to carcinogenic metabolites via the formation of the bay region diol epoxide. Purified recombinant human CYP1A1 and CYP1B1 exhibit higher BaP hydroxylase activities than recombinant CYP1A2, CYP2A6, CYP2C9, CYP2C19, and CYP3A4 enzymes (Shimada 2004). Using purified rat hepatic C Y P enzymes, C Y P l A l exhibited the highest catalytic activity toward hydroxylation of BaP at the 3 and 9 positions. Other enzymes such as CYP2C11, CYP2C6, CYP2B1, CYP1A2, and CYP2A1 also exhibit BaP hydroxylation activity but are less efficient (Ryan and Levin, 1990). Results of the present study show that MROD activity was similar in liver microsomes prepared from hypophysectomized rats and intact rats. D M B A treatment induced a 14-fold increase in M R O D activity in the intact rats and a 17-fold increase in M R O D activity in the hypophysectomized rats. Therefore, D M B A treatment elicited an increase in MROD activity, 89 which could be as a result of induction of hepatic CYP1A2 expression. According to our results, hepatic BaP hydroxylase activity was not affected by hypophysectomy in corn oil-treated female rats. A previous study reported on the effect of hypophysectomy and castration on hepatic BaP hydroxylase activity in male Sprague-Dawley rats (Al-Turk et al, 1981). That study showed a reduction in BaP hydroxlase activity in hepatic microsomes prepared from hypophysectomized male rats. However, our results demonstrated that hypophysectomy of female rats did not affect BaP hydroxylase activity. Because hydroxylation of BaP is mediated by several C Y P enzymes in uninduced rats, the difference in results between our study and that of Al-Turk et al. (1981) can be explained by a sex difference in C Y P enzyme expression in hypophysectomized male and female rats. Some hepatic C Y P enzymes such as CYP2C11 are expressed predominantly or solely in male rats, whereas other enzymes such as CYP2C12 are expressed only in female rats (Shapiro et al, 1995). Hepatic BaP hydroxylase activity was induced by D M B A treatment in hypophysectomized and intact rats. However, hepatic BaP hydroxylase activity was significantly higher in DMBA-treated intact rats than in DMBA-treated hypophysectomized rats. Several factors can affect the hydroxylation of BaP including inherited influences such as strain, age, C Y P enzyme expression and polymorphisms, and N A D P H cytochrome P450 reductase levels. BaP hydroxylation at the 3 or 9 position is mediated primarily by CYP1A1 (Ryan and Levin et al, 1989). According to the results obtained for hepatic CYP1A1 expression, there was no significant difference in the expression of hepatic CYP1 A l protein between DMBA-treated hypophysectomized and intact rats. N A D P H cytochrome P450 reductase, which catalyzes electron transfer from N A D P H to CYP, is an essential component for all CYP-mediated hydroxylation reactions. Although our study did not measure the 90 expression of N A D P H cytochrome P450 reductase in the liver, it was documented previously that hypophysectomy affected the expression and activity of N A D P H cytochrome P450 reductase in male and female Fischer rats (Waxman et al., 1989). However, MROD activity, which also requires the activity of N A D P H cytochrome P450 reductase, was similar between hypophysectomized and intact rats, suggesting a reduction in BaP hydroxylase activity is not due to altered N A D P H cytochrome P450 reductase level or activity. The results seen in the BaP hydroxylase activities could be affected by other factors such as the expression of other hepatic C Y P enzymes such as CYP2C and CYP2B which also contribute to the hydroxylation of BaP. The levels of these enzymes were not examined in this study. In our study, mEH enzyme activity was not examined. E H activity in hepatic microsomes can be determined by the radiometric method of Schmassmann et al. (1976) using [3H]benzo[a]pyrene-4,5-oxide as the substrate. Inoue et al., (1995) reported mEH expression and activity were induced by hypophysectomy in female mice. There was a 77% increase in mEH enzyme activity in hypophysectomized female mice compared against intact female mice. It was also reported previously that there is a sex-related and age-dependent difference in mEH activities in the Sprague-Dawley rats. Hepatic mEH activity demonstrated a developmental pattern in female rats, where mEH activity was low at the neonatal stage, followed by a rapid rise in activity at puberty and remained stationary for up to 90 days (Denlinger and Vesell, 1989). Male rats followed a similar age-development pattern in mEH activity, but the activity in adult male was significantly higher than adult female rats. Hormonal influences on the developmental pattern of mEH were also examined in that study. The administration of testosterone propionate to female rats increased hepatic mEH activity to a level similar to the adult male rats (Denlinger and Vesell, 1989). The data agree with our results and implicate a strong hormonal influence on both mEH expression and activity. 91 Additional experiments would be needed to identify the specific hormone or hormones that are involve in the regulation of mEH expression. 4.10 Evaluat ion of Hormones and D M B A - i n d u c e d M a m m a r y Tumorigenesis Hormones are an important factor in mammary gland development. Rat mammary gland expresses hormone receptors such as estrogen receptor, progesterone receptor, prolactin receptor, and also growth hormone receptors. Normal ductal branching and proliferation requires the presence of estrogen (Bocchinfuso and Korach, 1997) and progesterone (Humphrey et al., 1997) and an intact pituitary (Reece et al., 1936). It is evident that mammary gland growth is under the control of both the ovary and pituitary gland (see Figure 4.1). We speculate that the initiation and progression of DMBA-induced mammary cancer in rats depends on the interaction of multiple mammogenic and lactogenic factors. There have been numerous studies and reports investigating the role of ovarian hormones and pituitary factors on mammary tumorigenesis. Estradiol administered at low doses stimulates the growth of DMBA-induced mammary tumors (Huggins et al., 1958). However, the development and growth of these tumors are inhibited by high doses of 17P-estradiol (Huggins et al, 1958). Some researchers have hypothesized that estrogens bind to estrogen receptor to stimulate tumor growth (Lewis et al., 2004). Others have speculated that estrogens are mammogenic because of their stimulatory effects on prolactin secretion, where prolactin secretion is stimulated by low and inhibited by large doses of estrogens (Russo and Russo, 1998). Plaut et al. (1993) demonstrated that prolactin is essential for mammary lobulo-alveolar development in whole organ culture and that the mammary gland is more sensitive to prolactin than growth hormone in lobulo-alveolar development. The same study 92 also demonstrated that growth hormone does not mediate mammary gland development through binding to prolactin receptor nor is it mediated by IGF-1. Nevertheless, the mechanism of action of individual hormone in mammary tumor initiation and progression is still unclear. H y p o t h a l a m u s G o n a d o t r o p h i n R e l e a s i n g H o r m o n e P i t u i t a r y i r L H F S H J i O v a r y E s t r a d i o l P r o g e s t e r o n e " i r B r e a s t U t e r u s Figure 4.1 The pituitary releases gonadotrophins, luteinizing hormone (LH) and follicle-stimulating hormone (FSH) through the secretion of the gonadotrophin release hormone by the hypothalamus. The gonadotrophins then act on the ovary to stimulate the release of ovarian hormones, estradiol and progesterone to promote growth of the breast and uterus. 93 4.11 Summary 1. A single dose of 20 mg of D M B A caused a 66% mortality rate in the hypophysectomized rats. Deaths were related to the acute toxic effects of D M B A . When 20 mg of D M B A was given as four divided doses of 5 mg each, no deaths was observed in the hypophysectomized rats. 2. Hypophysectomized adult virgin female rats treated with a single intragastric dose of 20 mg of D M B A did not develop mammary tumorigenesis among the surviving rats, whereas 55% of the intact female rats treated with D M B A developed mammary tumors. Mammary tumor latency was approximately 8 weeks post D M B A treatment and there was an average of 2.5 tumors per tumor-bearing rat. 3. Basal hepatic CYP1A1 and CYP1A2 expression was unaffected by hypophysectomy. Hepatic C Y P 1-mediated activities measured by M R O D and BaP hydroxylase assays were also similar between intact and hypophysectomized rats. Mammary CYP1A1, CYP1A2, and CYP1B1 protein levels in corn oil-treated rats were below the detection limit as assessed by immunoblot analysis. D M B A treatment of intact and hypophysectomized rats induced hepatic CYP1A1 and CYP1A2 and mammary CYP1A1, CYP1A2, and CYP1B1 enzyme expression. Hepatic CYP 1-mediated enzyme activities were also increased in liver microsomes of animals treated with D M B A . 4. Hepatic and mammary mEH protein expression was significantly higher in hypophysectomized rats when compared to intact animals. D M B A treatment had no additional effect on hepatic and mammary mEH protein expression. The expression of 94 rat hepatic and mammary mEH appears to be regulated by pituitary hormones. 4.12 Conclusion and Future Directions Hypophysectomized rats failed to develop mammary tumors induced by D M B A . It was demonstrated in a previous study that tumors were induced by 3-MC in ovariectomized rats but at a decreased incidence compared to intact rats, whereas a complete failure of tumor induction was seen in hypophysectomized rats (Huggins et al, 1958). A factor or a combination of factors controlled by the pituitary gland appears to be an essential component in the initiation and development of breast cancer. The expression and activity of CYP1 and mEH enzymes needed for the bioactivation pathway of D M B A were measured between hypophysectomized and intact rats. Both CYP1 and mEH enzymes play important roles catalyzing the reactions that transform D M B A to the carcinogenic diol-epoxide metabolite. The formation of DMBA-3,4-diol, in particular, is limited by the availability of mEH (Christou et al., 1989). Although the present study did not directly measure D M B A bioactivation in intact and hypophysectomized rats, our results demonstrated that hypophysectomy did not affect the expression or induction of hepatic CYP1A1 and CYP1A2 or the expression of mammary CYP1A1, CYP1A2, and CYP1B1. Hepatic CYP1 enzyme activities in hypophysectomized rats were also measured and no significant difference was found between intact and hypophysectomized animals. Thus, the failure of hypophysectomized animals to develop mammary tumors following treatment with D M B A is not due to the bioactivation process of D M B A . Mammary tumorigenesis has a strong hormonal component. Estradiol, progesterone, growth hormone, and prolactin are well documented regulators of breast cancer growth. 95 Hypophysectomized rats failed to develop mammary tumors is most probable due to a suppression of ovarian and pituitary hormones. 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Proceedings of the National Academy of Sciences of the United States of America. 72: 2601-5. Yang, C S . and Kicha, L.P. (1978). A direct fluorometric assay of benzo[a]pyrene hydroxylase. Analytical Biochemistry. 84: 154-163. I l l 6. APPENDIX 6.1 Appendix I: Mortality in Hypophysectomized Rats Given Intermittent D M B A Doses 6.1.1 Introduction Study 1 had shown that hypophysectomized rats are more susceptible to the acute toxic effect of a single intragastric dose of 20 mg of D M B A (results shown in Table 3.3). Since the acute toxic effects of D M B A are likely to be dose-dependent, administering lower amounts of D M B A with intermission may reduce mortality while conserving the carcinogenic effects. In this experiment, we proposed a D M B A dosing regimen similar to the method described by Hollingsworth et al. (1998) to induce mammary tumors in hypophysectomized rats. 6.1.2 Experimental Design Fourteen hypophysectomized rats were treated with four doses of 5 mg of D M B A dissolved in 1 ml of corn oil (a total of 20 mg of D M B A ) over a 2-week period. The timing and interval of each dose is shown in Figure 6.1. Body weights of the animals were measured daily during the study period. A l l deaths were recorded. The animals were terminated by decapitation 24 hours after the last D M B A dose. 112 D M B A treatment Termination 1 s t dose J 2 n d dose J 3 r d dose J 4 t h dose J 14 Time (Days) Figure 6.1 Experimental Design: Mortality in hypophysectomized rats given intermittent D M B A doses. 6.1.3 Effect of Intermittent DMBA Treatment on Body Weight Body weight was monitored daily and throughout the treatment period. The daily change in body weight is illustrated in Figure 6.2. Similar to the results seen in Study 1, hypophysectomized rats demonstrated little or no weight gain during the monitoring period. The animals had an average body weight of 136 ± 12 g in the beginning of the study and an average of 149 ± 36 g at termination. Between each 5 mg dose of D M B A , no significant weight loss was observed in the animals. 6.1.4 Effect of Intermittent DMBA Treatment on Mortality The results of Study 1 demonstrated that more than 66% of hypophysectomized rats died after a single intragastric dose of 20 mg of D M B A . In contrast, no deaths were observed in the hypophysectomized rats given 20 mg of D M B A as intermittent doses. Hypophysectomized rats were able to tolerate 20 mg D M B A administered as four divided doses of 5 mg each. 113 180 160 140 120 £ 100 O) "53 >, 80 TO o m 60 40 20 0 1st dose 2nd dose 3rd dose 4th dose 22-Jan 25-Jan 28-Jan 31-Jan 3-Feb 6-Feb 9-Feb 12-Feb 15-Feb 18-Feb 21-Feb 24-Feb 27-Feb 2-Mar 5-Mar Date Figure 6.2 Average body weight of rats during the experimental period {n = 14). Values are expressed as mean ± SEM. 

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