© 2000 by Oxford University Press
Journal of the National Cancer Institute Monographs, No. 27, 95-112,
2000
© 2000 Oxford University Press
Chapter 5: Tissue-Specific Synthesis and Oxidative Metabolism of Estrogens
Affiliations of authors: C. R. Jefcoate, Department of Pharmacology, University of Wisconsin—Madison; J. G. Liehr, Stehlin Foundation for Cancer Research, Houston, TX; R. J. Santen, W. Yue, Division of Hematology, Oncology, and Endocrinology, Cancer Center, University of Virginia Health Science Center, Charlottesville; T. R. Sutter, W. Harry Feinstone Center for Genomic Research, University of Memphis, TN; J. D. Yager, Division of Toxicological Sciences, Department of Health Sciences, The Johns Hopkins University School of Hygiene and Public Health, Baltimore, MD; S. J. Santner; R. Pauley, Wayne State University, Detroit, MI; R. Tekmal; Emory University, Atlanta, GA; L. Demers, Pennsylvania State University, Hershey; F. Naftolin, G. Mor, Yale University, New Haven, CT; L. Berstein, Petrov Institute, St. Petersburg, Russia.
Correspondence to: James D. Yager, Ph.D., Division of Toxicological Sciences, Department of Environmental Health Sciences, The Johns Hopkins University School of Hygiene and Public Health, 615 North Wolfe St., Baltimore, MD 21205 (e-mail: jyager{at}jhsph.edu).
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ABSTRACT |
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 TopNotes Abstract IntroductionPotential Role of Aromatase...Metabolic Activation of...Estrogen 4-Hydroxylation...Identification and...Estrogen Hydroxylation Catalyzed...References |
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Estrogen exposure represents the major known risk factor for development of breast cancer in women and is implicated in the development of prostate cancer in men. Human breast tissue has been shown to be a site of oxidative metabolism of estrogen due to the presence of specific cytochrome P450 enzymes. The oxidative metabolism of 17
-estradiol (E2) to E2–3,4-quinone metabolites by an E2-4-hydroxylase in breast tissue provides a rational hypothesis to explain the mammary carcinogenic effects of estrogen in women because this metabolite is directly genotoxic and can undergo redox cycling to form genotoxic reactive oxygen species. In this chapter, evidence in support of this hypothesis and of the role of P4501B1 as the 4-hydroxylase expressed in human breast tissue is reviewed. However, the plausibility of this hypothesis has been questioned on the grounds that insufficient E2 is present in breast tissue to be converted to biologically significant amounts of metabolite. This critique is based on the assumption that plasma and tissue E2 levels are concordant. However, breast cancer tissue E2 levels are 10-fold to 50-fold higher in postmenopausal women than predicted from plasma levels. Consequently, factors must be present to alter breast tissue E2 levels independently of plasma concentrations. One such factor may be the local production of E2 in breast tissue through the enzyme aromatase, and the evidence supporting the expression of aromatase in breast tissue is also reviewed in this chapter. If correct, mutations or environmental factors enhancing aromatase activity might result in high tissue concentrations of E2 that would likely be sufficient to serve as substrates for CYP1B1, given its high affinity for E2. This concept, if verified experimentally, would provide plausibility to the hypothesis that sufficient E2 may be present in tissue for formation of catechol metabolites that are estrogenic and which, upon further oxidative metabolism, form genotoxic species at levels that may contribute to estrogen carcinogenesis.
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INTRODUCTION |
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TopNotesAbstractIntroductionPotential Role of Aromatase...Metabolic Activation of...Estrogen 4-Hydroxylation...Identification and...Estrogen Hydroxylation Catalyzed...References |
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The major known risk factors for development of breast cancer in women are associated with prolonged exposure to increased levels of estrogen. Estrogen and testosterone are also thought to be involved in the development of prostate cancer in men (see Chapter 2). From information presented in Chapters 3 and 4, it is clear that evidence is building for a role of oxidative metabolites of 17
-estradiol (E2) and/or estrone (E1), particularly the catechols, in breast cancer. New information implicates the catechols as signaling molecules with relative binding affinities for the human estrogen receptor that are equal to or greater than E2 itself (1). It is also clear that, upon further oxidative metabolism, the catechol metabolites can form quinones that can directly form adducts with glutathione and guanine and adenine bases in DNA (see Chapter 4). In particular, the 3,4-quinone forms a depurinating adduct with guanine and adenine, leaving an abasic site with mutagenic potential. In addition, as will be discussed in more detail below, the catechols are capable of redox cycling, a process accompanied by the generation of reactive oxygen species able to cause oxidative damage to lipids, proteins, and DNA. A critical issue in relation to estrogen and the potential contribution of the catechol estrogen (CE) metabolites to breast cancer is their source. Estrogens themselves and their oxidative metabolites are formed by the activities of various cytochromes P450 (CYPs). These enzymes have dual functions, the biosynthesis and/or inactivation of physiologic regulators on the one hand and the metabolism of environmental chemicals on the other. Natural processes in which they participate include the synthesis of estrogen from cholesterol, which involves multiple, very specific CYP enzymes, typically compartmentalized into different organelles, cells, or organs. The final process in the synthesis of estrogens involves a three-step oxygenation reaction catalyzed by a single P450, CYP19 (aromatase), which converts an androgen (testosterone or androstenedione) to an estrogen (E2 or E1, respectively). Numerous low-specificity CYPs are involved in the oxidative inactivation and clearance of these same steroids, drugs, and environmental pollutants. The low specificity of these P450 cytochromes, which are most abundant in liver but are also found in most cells, results in the conversion of these chemicals to multiple products with increased hydrophilicity and functional groups for subsequent metabolism. For some chemicals, particularly those that contain olefinic double bonds or aromatic rings, these products may include chemically reactive metabolites that can cause DNA damage and thereby cause errors during replication, which result in mutations. The metabolism of steroids, in conjunction with conjugation reactions (sulfation, glucuronidation, and methylation), may contribute to lowering serum and cellular levels of steroidal parent hormones and, hence, to altered regulation of biologic activity. Identification of any new P450 cytochrome raises the question of which of these functions underlie its pattern of expression. It is most important to appreciate that the various P450 enzymes show tissue-specific expression; i.e., forms expressed in liver may not reflect those expressed in breast or prostate tissue. The implications of this are potentially enormous, since metabolites (whether of exogenous or endogenous compounds) will show tissue specificity.
The mammary gland is involved in steroid synthesis (certainly aromatase and possibly other steps), steroid metabolism (hydroxylation and conjugation), and xenobiotic metabolism (lipophilic molecules stored in fat and excreted in the milk). As will be discussed in detail below, an investigation (2) indicated that substantial levels of estrogen arise from aromatase activity localized in breast tissue. This is present in both breast epithelia and fibroblasts. Thus, estrogen biosynthesis occurs in the target tissue. Furthermore, the estrogen oxidative metabolites are formed by various CYPs. Human breast and breast tumor tissue express various CYPs. Among these is CYP1B1. As will be discussed in detail below, CYP1B1 is a catalytically efficient estrogen 4-hydroxylase. CYP1B1 is present in fibroblasts, as well as in epithelial cells (3–6); thus, it is co-localized with aromatase, the enzyme producing estrogens. This co-localization of CYP1B1 and aromatase means that the effective concentration of the 4-hydroxylated product is much higher than that indicated by the circulating levels. Human breast fibroblasts contain estrogen receptors that regulate cell growth in vitro, but estrogen receptors are present in only 5%–10% of the epithelial cells. A study (7) indicated that aromatase is elevated by prostaglandin E2 (via cyclic adenosine monophosphate [cAMP]) and by various cytokines. However, very little is known about the regulation of CYP1B1 and estrogen receptors in these cells. Expression of each of these genes is also dependent on the extracellular matrix and growth factor milieu that surrounds these cells. Several presentations in this monograph raise the issue of oxidative stress (see Chapters 3 and 4), mediated by the 4-CE/o-quinone redox cycling. One possibility is that this may also contribute to signaling that promotes aromatase expression. The remainder of this chapter will provide greater detail regarding the expression and potential roles of aromatase, CYP1B1, and CE, particularly 4-hydroxy-E2 (4-OHE2), in breast cancer.
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POTENTIAL ROLE OF AROMATASE OVEREXPRESSION IN THE GENESIS OF BREAST CARCINOMA |
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TopNotesAbstractIntroductionPotential Role of Aromatase...Metabolic Activation of...Estrogen 4-Hydroxylation...Identification and...Estrogen Hydroxylation Catalyzed...References |
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Estrogens and Risk of Breast Cancer
Evidence from multiple sources suggests that estrogens are involved in the genesis of breast carcinoma. Administration of exogenous estrogens causes breast cancer in rodents (8). Breast tumors induced by the potent carcinogen 7,12-dimethylbenz[a]anthracene can be delayed or prevented by castration and administration of anti-estrogens (9). Aromatase inhibitors prevent the spontaneous development of breast tumors in aging Sprague-Dawley rats (10). By the age of 2 years, approximately 50% of these animals develop either benign or malignant breast lesions (Fig. 1
) (10). Increasing doses of the aromatase inhibitor fadrozole inhibit these tumors in a dose-dependent fashion. Notably, 1.25 mg of fadrozole/kg of body weight daily causes 100% inhibition. Taken together, these data provide strong evidence that estrogens are involved in the carcinogenic process, resulting in breast neoplasms in animals.
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Indirect evidence in women also supports a role for estrogen in the genesis of breast cancer. Early menarche and late menopause are associated with an increased risk of breast cancer. These factors result in prolonged exposure of the breast to estrogen. Prospective epidemiologic studies (11–14) detected an increase in the plasma levels of E2 in women developing breast cancer 5 or more years later. Obesity is also associated with a greater risk of breast cancer (15). This relationship might also be explained by increased estrogen production, since the degree of obesity correlates linearly with total-body aromatase activity (16). Aromatase catalyzes the rate-limiting step in estrogen biosynthesis, the conversion of androgens to estrogens.
Additional indirect evidence regarding estrogens and breast cancer derives from analyses of the rate of breast cancer in women receiving estrogen replacement therapy during the menopause. More than 50 studies are now available to examine this relationship [reviewed in (17)]. Six meta-analyses have pooled this information, and a critical review of these data allows several points to be made. The first is that no randomized, controlled, double-blind studies have been conducted to demonstrate conclusively that estrogen replacement therapy during the menopause increases the risk of breast cancer. Only observational studies are available. Secondly, while susceptible to several biases, most of these studies show an increased risk of breast cancer with the use of estrogen replacement therapy for a period of more than 5–10 years. The relative risk of breast cancer under these circumstances increases by about 30%. Thirdly, the absolute increased risk is small, approximating one additional breast cancer case in 100 women of age 50 years who have taken estrogen for at least 10 years.
The conclusion that these data prove that estrogen replacement therapy is associated with an increased risk of breast cancer is controversial. However, as current studies evolve, the evidence increases. For example, the Nurses' Health Study (18) now involves more than 725 550 patient-years of observation over a 10-year period. In this study, the risk of breast cancer increased with more than 5 years of current estrogen use in women of all ages over 50 years. This cohort study allows adjustment for most, but not all, confounding factors and provides the most convincing evidence of a relationship between estrogen ingestion and breast cancer. The definitive study, conducted as part of the Women's Health Initiative, involves a randomized, placebo-controlled trial. The results of this study will not be available until midway into the first decade of the 21st century.
Correlations between estrogen levels and subsequent risk of breast cancer have not, until recently, been positive. Earlier studies measured levels of urinary estrogens, plasma estrogens, or the free fraction of E2 with insensitive or relatively nonspecific methods. Conflicting results were reported, and the common view was that estrogens were not elevated in women who would later develop breast cancer (19,20). Recently, however, Toniolo et al. (11) and three other groups (12–14) demonstrated increased levels of E2 and its precursor, testosterone, in women found to develop breast cancer prospectively 5 or more years later. While not every group could replicate these results (19,20), the majority of data support such a relationship.
Further evidence regarding estrogen production and breast cancer risk has been provided by experiments demonstrating that a reduction in estrogen production in women reduces the incidence of breast cancer (21,22). Data from two classic studies (21,22) are consistent with such an effect (Fig. 2). One of these studies (22) examined the incidence of breast cancer in a group of women who had undergone bilateral oophorectomy before age 35 years. The control group consisted of women subjected to a unilateral oophorectomy at the same ages. The end point of the study was the ratio between observed and expected breast cancers in these two groups of women. After a period of 20 years, the women undergoing bilateral oophorectomy had a 75% reduction in the incidence of breast carcinoma (Fig. 2, A). In the other study (21), with a similar design, the decrease in breast cancer incidence over that expected gradually declined as a function of time after oophorectomy (Fig. 2, B). Although these studies were also subject to bias, they provide compelling evidence that ovarian factors, and presumably E2, are involved in the genesis of breast carcinoma.
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Sources of Estrogen
The sources of E2 production in women are important to consider, since overproduction may result from altered regulation at any site. Estrogen can be made in several tissues. Aromatase, the enzyme catalyzing the rate-limiting step in estrogen biosynthesis, is widely present throughout the body. The premenopausal ovary, which contains the highest level of aromatase, except for the placenta, is the major source of E2 during the premenopausal years. Peripheral adipose tissue also contains aromatase and is a major source of this enzyme, since the mass of adipose tissue (particularly in obese women) is substantial. Breast tissue itself contains aromatase, both in its fatty components and in its epithelial cells, and can synthesize estrogen in situ.
Importance of In Situ Aromatase in Breast Tissue
Emerging evidence suggests that estrogen produced in situ, as opposed to E2 made in other tissues and delivered to the breast via an endocrine mechanism, plays a major biologic role in breast physiology. Several lines of evidence support this concept. Demonstration of the aromatase enzyme and its messenger RNA in breast tissue by immunohistochemical and molecular biologic techniques, studies in nude mice to show that the amount of estrogen made locally causes biologic effects, and clinical studies of aromatase inhibitors in patients provide proof of the importance of in situ production of estrogen in breast tissue.
Several groups of investigators (23–25) over the past few years have demonstrated the aromatase enzyme in breast tissue. Both radiometric and product isolation methods demonstrated that tritiated androgens could be converted to estrogens in human breast cancer tissue as evidence of aromatase activity. The biologic significance of this finding was initially questioned, since the amount of enzyme present, compared with that in placenta or ovary, was low (26). In this regard, other investigators postulated that focal concentrations of aromatase in selected cell populations might be high, but overall activity in breast cancer tissue might be low. This might occur because of the presence of fibrous or other tissues in the tumor, which would dilute the concentration of enzyme in the tumor overall. To examine this possibility, immunohistochemical studies were used to detect aromatase in individual breast cancer cells. Resulting data (27–29) demonstrated high levels of aromatase staining in individual cells, supporting the concept that aromatase might act in an autocrine or paracrine fashion in breast tissue (Fig. 3).
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Controversy exists at present whether aromatase activity is predominantly in epithelial cancer cells or in the surrounding stromal cells (30,31). Certain monoclonal antibodies, used in conjunction with antigen retrieval techniques, suggest that the majority of aromatase is in epithelial cells (30). Monospecific polyclonal antibodies, utilized on the same tissue sections, show a preponderance of aromatase activity in stromal cells (27,32,33). To provide additional data regarding stromal aromatase, Santen and his group (33) grew stromal cells isolated from breast cancer lesions as well as from benign tissue surrounding the tumors. They found that isolated stromal cells from breast cancer tissue contain high levels of aromatase enzyme when stimulated by dexamethasone, phorbol esters, and cAMP in combination. Aromatase enzyme activity, assessed by a radiometric assay, increases by nearly four logs in response to this combination of enhancers, and message increases 30-fold (Fig. 4
). Other investigators have found that aromatase message levels are higher in areas of breast cancer with high stromal cell content than in areas with low content. Quantitative assessment using a histologic scoring system and immunohistochemistry detected average H-scores of 13 (range, 0–45) for stromal cells and 4 (range, 1.4–16) for stroma in 26 human breast tumors (27). This technique takes into account the relative abundance of stromal and epithelial cells in the tumor (27). Considered together, these data support the biologic importance of aromatase in breast cancer tissue and suggest that stroma may contribute to a greater extent to this process than epithelial cells.
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Further support for the importance of aromatase in breast tissue itself derives from studies in a nude mouse model developed by Yue et al. (34). Using this model, these investigators (34) examined the relative importance of uptake from plasma versus local E2 synthesis in breast tissue. This model involves the use of MCF-7 breast cancer cells transfected stably with aromatase (A+) that are implanted on one side of castrated nude mice. On the other side, sham-transfected MCF-7 cells (A-) are implanted. Administration of the aromatase substrate androstenedione causes no growth stimulation of aromatase-negative cells (Fig. 5
). This important control demonstrates that no aromatase activity is present in non-breast tissue in the mouse. Aromatase-positive cells implanted on the other side of the same animals are stimulated to grow by androstenedione, providing evidence of the biologic effect of aromatase present locally in the breast. The aromatase inhibitor 4-hydroxyandrostenedione blocked this growth effect. One can conclude that the growth stimulation cannot have occurred as a result of peripheral conversion to E2, which would have stimulated the aromatase-negative cells as well. As expected, the levels of E2 in aromatase-positive tumors markedly exceeded those in aromatase-negative tumors (Fig. 6).
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The relative importance of in situ production versus uptake of E2 from plasma was then examined. Silastic implants designed to produce plasma estrogen levels ranging from 5 to 20 pg/mL were implanted into castrate animals to evaluate the effect of E2 uptake. Androstenedione was administered to others to examine in situ production. With this experimental system, tissue E2 levels and tumor growth were higher as a result of in situ aromatization than from plasma delivery of estrogen [data shown in (34)].
This series of experiments in mice supports the hypothesis that an important determinant of tissue E2 levels is local production in the breast. If this hypothesis is correct, the level of E2 produced in breast tissue may be the most important determinant of E2-induced carcinogenesis. This conclusion is supported by the direct measurement of aromatase activity with elegant isotopic techniques in human breast tumors by Reed et al. (35). These investigators demonstrated that 83% ± 9% (standard deviation) of tumor estrogen levels resulted from in situ aromatase in four of six tumors. In the other two tumors, no estrogen synthesis in the breast itself could be demonstrated. Although speculative, the hypothesis of in situ E2 synthesis could explain the relatively poor correlations between use of estrogens for menopausal replacement therapy and breast cancer risk. This might also explain why it has been difficult to demonstrate higher estrogen levels in women who will later develop breast cancer. If the local synthesis hypothesis is correct, measurement of the concentration of E2 in breast tissue itself would be the most precise predictor of later development of breast cancer. This concept is supported by the fact that plasma and tissue E2 levels are not well correlated. The ratio of tissue-to-plasma E2 levels in premenopausal women approximates 1 : 1, whereas in postmenopausal women the ratio is 10–50 : 1 (36). Taken together, these data suggest that certain factors present in breast tissue can influence local production of estrogen and that these may be the prime determinants of tissue estrogen concentrations. If these concepts are correct, elevated plasma levels of estrogen would be associated with high tissue concentrations in some, but not in all, patients and breast cancer risk might only be increased in those with high tissue levels.
Mechanism of Carcinogenesis
After considering the sources of E2 available for stimulating breast tissue, one must consider how E2 causes breast cancer. The predominant theory at the present time relates to effects of estrogen on cell growth (37). Enhanced cell proliferation, induced either by endogenous or by exogenous estrogens, increases the number of cell divisions and, by inference, the proportionate number of mutations. With an enhanced rate of proliferation, the time available for DNA repair is reduced. In addition, single-stranded DNA, present during cell division, is more susceptible to damage than double-stranded DNA, and gene duplication can occur.
Another current theory, discussed in Chapters 3 and 4 and in the following section, is that estrogens can be metabolized to genotoxic products. These two current theories of enhanced cell proliferation and genotoxic metabolites are not mutually exclusive but could act in an additive or even synergistic fashion. For example, DNA damage originating from CEs would be propagated more rapidly by increased cellular proliferation, and insufficient time might be available for DNA repair. Additional data will be required to determine the precise interactions between these two pathways of carcinogenesis.
The major critique of the genotoxic metabolite theory is that estrogen levels are not sufficiently high to produce biologically relevant amounts of these metabolites. This critique, however, is based on an analysis of plasma E2 levels and not tissue levels. If E2 is synthesized locally in breast tissue, the levels would be higher than expected from plasma concentrations. This concept is supported by the fact that E2 concentrations in breast cancers from postmenopausal women are as high as those from premenopausal women. This is surprising, since the levels of E2 in the plasma of premenopausal women are 10- to 50-fold higher than those in the plasma of postmenopausal women (36).
Hypothesis of Aromatase Overexpression
As a means of integrating these data, Santen et al. (personal communication) have postulated that aromatase overexpression in breast tissue may be a cause of breast cancer. Through aromatase overexpression, tissue levels of E2 would be sufficiently high to undergo metabolism to biologically important quantities of genotoxic metabolites. Four separate models of aromatase overexpression and breast cancer have been well characterized and provide strong support for this hypothesis. Three involve the hyperplastic alveolar nodule (HAN) model systems. Zhang and Medina (38) have developed a series of transplantable breast explants that grow in the mammary fat pads of highly inbred strains of mice. Two of these are induced by carcinogens and are called the C4 and C5 HAN. One is induced by hormonal stimulation and is called the D2 HAN. Upon serial passage in mammary fat pads, these lesions develop frank cancer with an incidence that approaches 90% under certain conditions. Each of these HAN models has been shown to have an insertional mutation called Int 5. This mutation has now been characterized and involves the insertion of a portion of the long terminal repeat of the mouse mammary tumor virus into genomic DNA (39–41). Of great interest is the fact that, in each of these models, the insertion is into the 3` untranslated region of the tenth exon of the aromatase gene. This results in overexpression of the aromatase gene and, by inference, in tumor development. The fourth model is a transgenic mouse model in which aromatase is overexpressed, predominantly in mammary tissue (42). These animals develop atypical ductal hyperplasia, a type of lesion that predicts an increased rate of breast cancer development when found in patients. They also develop fibroadenomas, typical ductal hyperplasia, and dysplasia, lesions that are not associated with an increased risk of breast cancer in women. Rarely do the aromatase-transfected animals develop frank breast cancer.
Evidence of Aromatase in Benign Breast Tissue
Several laboratories have obtained evidence that malignant breast tissue contains both aromatase message and enzyme activity. However, if aromatase overexpression is important in the genesis of breast cancer, this enzyme must be present in benign breast tissue as well. To assess this possibility, core and excisional biopsy specimens containing atypical ductal hyperplasia were evaluated with an immunohistochemical method using a monospecific polyclonal antibody (43). This technique revealed aromatase immunohistochemical staining in both stromal and epithelial cells contained in the hyperplastic lesions. In the surrounding normal tissue, aromatase staining was present predominantly in glandular epithelial cells but to a lesser extent in stroma.
Surprisingly, macrophages with substantial aromatase activity were also detected (43). This finding led to an extensive series of experiments to verify that macrophages indeed express aromatase. THP-1 cells, a malignant cell line that can be differentiated into macrophages upon exposure to phorbol esters (44), were used. These cells contained aromatase enzyme activity with levels close to those found in human placenta. The aromatase inhibitor letrozole completely inhibited this activity. Conditioned media from these cells, exposed to the aromatase substrate testosterone, stimulated the growth of E2-responsive MCF-7 indicator cells. As evidence of specificity, growth of indicator cells could be blocked with letrozole or with the pure anti-estrogen ICI 182780.
As further evidence of aromatase expression in macrophages, human monocytes were examined, basally and after differentiation into macrophages, in tissue culture with the addition of phorbol esters. These cells contained aromatase message when differentiated into macrophages but not under control conditions. Finally, monoclonal antibodies specific for macrophages were used to demonstrate, by double labeling, that the cells in the breast that contained aromatase were in fact macrophages.
The production of E2 by macrophages is of further interest because chemokines regulating tissue invasion by macrophages are also controlled by estrogen. The levels of macrophage chemokine 1 (MCP-1) are lowered by E2 in MCF-7 breast cancer cells and in other tissues (see Chapter 8). It is interesting that a classical negative feedback loop could exist whereby E2 lowers MCP-1, which would result in a decrease in invasion of tissue by macrophages. This would result in a lower production of E2 in that tissue. As a consequence, the levels of MCP-1 would increase and the macrophages would again be stimulated to invade the tissue. Since breast tumors contain a substantial number of macrophages, their contribution to local E2 production could be biologically important.
These data suggest that breast tissue can make E2 from epithelial cells, from stroma, and from macrophages that infiltrate normal tissue. Potentially, either one of these three cell types could overexpress aromatase and provide sufficient amounts of E2 locally to allow conversion to genotoxic quinone metabolites. Several examples of aromatase overexpression are known to exist. A rat Leydig cell tumor overexpresses aromatase through activation of a cAMP-dependent enhancer of aromatase (45). The breast tissue of goats is the major source of aromatase prior to parturition, and bilateral mastectomy delays the time of parturition (46). The Sebright bantam syndrome is caused by aromatase overexpression, which feminizes the feather pattern of roosters and gives them the phenotypic appearance of chickens (47). Familial causes of aromatase overexpression occur in patients, resulting in prepubertal gynecomastia in boys and precocious thelarche and/or macromastia in girls. The Peutz-Jeghers syndrome is characterized by aromatase overproduction and leads to testicular tumors in boys and ovarian tumors in girls. Each of these examples provides evidence that aromatase overexpression is somewhat common in animals and in patients (48).
Mechanism of Aromatase Overexpression
A variety of potential mechanisms could result in aromatase overexpression. Aromatase transcription is regulated by multiple enhancers, including cAMP, phorbol esters, dexamethasone, prostaglandin E2, transforming growth factor- and interferon gamma among others (49). Fibroblasts isolated from breast tumors as well as from benign tissue surrounding the tumors contain aromatase. The activity of this enzyme and its message can be stimulated up to 10 000-fold in cell culture with addition of phorbol esters, cAMP, and dexamethasone (Fig. 4
) (41). Activating mutations involving any of these or other steps could result in aromatase overexpression in breast tissue. Simpson and co-workers (50) have postulated that prostaglandin E2 may be important in this process and have pointed out that use of nonsteroidal anti-inflammatory agents is associated with a decreased incidence of breast cancer in women. These agents are known to block prostaglandin E2 production and putatively could decrease breast cancer through this mechanism.
Prevention of Breast Cancer
If aromatase overexpression were an etiologic factor for breast cancer, third-generation aromatase inhibitors might be used for prevention. In premenopausal women, it would be possible to block estrogen production in breast tissue without affecting ovarian E2 synthesis. The ovary is relatively resistant to aromatase inhibitors because of the extremely high levels of androstenedione present as substrate in the ovary (51). While these concepts are speculative, further evaluation of aromatase expression in various premalignant breast lesions is warranted.
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METABOLIC ACTIVATION OF ESTROGENS BY 4-HYDROXYLATION |
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TopNotesAbstractIntroductionPotential Role of Aromatase...Metabolic Activation of...Estrogen 4-Hydroxylation...Identification and...Estrogen Hydroxylation Catalyzed...References |
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Role of Metabolism in Estrogen-Induced Cancer
The oxidative metabolism of estrogens has been studied in detail as part of investigations of the regulation and control of hormonal action and has been reviewed by Zhu and Conney (52). In this section, estrogen metabolism will be discussed only to the extent that it affects induction of tumors by this hormone. The metabolic activation of estrogens has increasingly been suspected to play a role in the carcinogenic process (53–55) because the modulation of metabolic oxidation affects E2-induced tumor incidence in a rodent model, the kidney tumor in male Syrian hamsters, in a way that is not consistent with previous hypotheses. Estrogens have previously been postulated to act in hormone-associated cancers, including breast cancer, primarily by estrogen receptor-mediated proliferation of cells mutated by spontaneous replication errors [reviewed by Feigelson and Henderson (56)]. According to this hypothesis, inhibition of estrogen metabolism should enhance tumor formation, since estrogen metabolites are less hormonally active than the parent E2 or E1. In contrast, 
-naphthoflavone, an inhibitor of estrogen hydroxylation (57,58), and ascorbic acid (vitamin C), a reducing agent known to reduce estrogen quinone intermediates to their hydroquinones (59), either completely or partially inhibited kidney tumor induction in hamsters by E2 (Table 1) (59–61). Neither of these chemicals is known to have estrogen agonist or antagonist activities and, thus, could not have interfered with the estrogen receptor-mediated proliferation of mutated cells. In addition, 17-ethinylestradiol, a poor carcinogen in the hamster kidney tumor model (62), is converted to catechol metabolites at much lower rates than E2 (63). All of these data are consistent with the conclusion that metabolic activation of estrogens is necessary for the induction of tumors by estrogenic hormones (53–55). This metabolic activation of estrogens, as a necessary part of the tumorigenesis process, has been proposed by analogy to metabolic activation of carcinogenic chemicals such as benzo[a]pyrene or other carcinogenic hydrocarbons (64). Moreover, the conversion to catechol metabolites and their further metabolism to quinone and semiquinone intermediates were explored, since such metabolites have been shown to play a role in DNA damage induced by other carcinogens (65).
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Formation of CEs
CEs, 2- or 4-hydroxylated E2 or E1, are the focus of metabolic research in the context of estrogen-induced cancer because these compounds are major oxidized metabolites of estrogenic hormones in most mammalian species (66,67) and are precursors to reactive intermediates (53–55). For instance, catechols including CEs may be oxidized chemically or by enzymatic processes to semiquinones, which are free radicals, and further to reactive quinone intermediates (68–71). These semiquinone/quinone species react with nucleophilic sulfur- or nitrogen-rich endogenous chemicals including nucleic acids (71–74). Therefore, the formation and metabolic activation of CEs, specifically 4-hydroxylated estrogens, are described below.
Catechol Formation by Aromatic Oxidation of Estrogens
2-Hydroxylation of steroidal estrogens is the major metabolic oxidation of estrogenic hormones in most mammalian species (Fig. 7) (66,67). For instance, in human or hamster livers, the 2-hydroxylation is catalyzed by CYP 3A isoforms, whereas CYP 1A isoforms represent the predominant estrogen-2-hydroxylase activity in extrahepatic tissues (57,75–77). These estrogen-2-hydroxylases convert E2 to approximately 80%–85% 2-hydroxyestradiol (2-OHE2) and, because of a lack of specificity of the enzyme(s), 15%–20% of 4-OHE2 (78), as shown, for instance, for liver in Table 2. In contrast, specific estrogen-4-hydroxylase(s) that convert E2 mainly to 4-OHE2 (3) have been identified (78–80) in those organs of rodents in which chronic estrogen exposure induces malignant or benign tumors, such as in hamster kidney (62), mouse uterus (81), or rat pituitary (82). The specific formation of 4-hydroxylated estrogens is important because, in the hamster kidney tumor model, 4-OHE2 is as carcinogenic as E2, whereas 2-hydroxylated estrogens did not induce any tumors (83–85). In addition to the specific reduced nicotinamide adenine dinucleotide phosphate (NADPH)-dependent estrogen 2- or 4-hydroxylation, an organic hydroperoxide-dependent estrogen 2- and 4-hydroxylase activity has been detected, which produces both catechols in roughly equal amounts (78,86).
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In humans, the predominant conversion of E2 to 4-OHE2 has been detected in microsomes of uterine myometrium and fibroids—i.e., benign uterine myomas (87)—and in benign and malignant mammary tumors (88). In addition, a specific estrogen-4-hydroxylase activity has been identified in MCF-7 breast cancer cells, which can be induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a common environmental pollutant (89,90). As described in the introduction to this chapter, this human estrogen-4-hydroxylase activity was designated as cytochrome P4501B1 (CYP1B1), a novel extrahepatic isozyme detected in mammary tissue, ovary, adrenal gland, uterus, and elsewhere (89–91). In one reported measurement of estrogen metabolite concentrations in a human breast cancer extract (92), the ratio of 4-OHE2 to 2-OHE2 was determined to be 4 : 1. The same 4 : 1 ratio was detected for the rates of formation of these CEs by breast cancer microsomes (88). It was concluded from these studies that, in rodent organs prone to estrogen-induced cancer or in human uterus or breast, which are targets of hormone-associated cancers, the predominant formation of 4-OHE2 may result in elevated concentrations of this carcinogenic estrogen metabolite in these tissues.
Catechol Formation by Deconjugation of Conjugated Estrogens
De novo formation of CEs by hydroxylation of E1 or E2 by CYP enzymes (66,67) is only one of several pathways of CE formation. Other pathways include the deconjugation of conjugated estrogen metabolites. The demethylation of methoxyestrogens has been investigated with the use of liver and kidney microsomes of male Syrian hamsters (93). Rates of demethylation of 2- and 4-methoxyestradiol by kidney microsomes are comparable, whereas the rate of demethylation of 2-methoxyestradiol by liver microsomes is approximately fivefold higher than that of 4-methoxyestradiol (Table 3) (93). The rates of renal demethylation of methoxyestrogens are comparable to the rates of direct 2- and 4-hydroxylation of E2 by kidney microsomes, whereas the rates of hepatic demethylation are approximately one fifth of the corresponding de novo hydroxylation rates (78). These data demonstrate that metabolic demethylation of methoxyestrogens is an important source of CE metabolites in the hamster kidney, a target of estrogen-induced carcinogenesis (62), whereas CE formation by direct aromatic hydroxylation of E2 predominates in liver, where this hormone does not induce tumors under these conditions. The identities of the CYP enzymes catalyzing demethylation of methoxyestrogens are not known. CYP 3A enzymes have been identified as one of the activities capable of catalyzing the N-demethylation of a xenobiotic substrate (94). Increased demethylase activity for methoxyestrogens in liver microsomes from phenobarbital-treated rats also indicates participation of CYP 2B isoforms in this reaction (95). However, specific identification of the enzymes catalyzing this reaction requires further research.
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Other phase II estrogen metabolites, such as E2- and E1-3-D-glucuronides, are also deconjugated to the parent hormones by lysosomal glucuronidases (93). For instance, lysosomes from male Syrian hamster kidney catalyze the deconjugation of estrogen glucuronides at rates that are one-third to two-thirds greater than corresponding rates by liver lysosomes. Treatment of hamsters with E2 implants for 9 days increases lysosomal glucuronidase activities for these estrogen glucuronides by 15%–25% in kidney and doubles the activities in liver, but it does not alter their corresponding Km (i.e., Michaelis constant) values (93). Microsomal glucuronidase activities are approximately 10%–20% of lysosomal activities and do not appear to contribute appreciably to deconjugation of glucuronide metabolites. These results have been obtained with E2- and E1-3-D-glucuronides as substrates. However, CE-glucuronide metabolites exist (96) and may also be deconjugated by these enzymes. Therefore, conjugates of both CE metabolites and of parent hormones formed in the liver may circulate and be deconjugated in extrahepatic tissues, including in organs prone to hormonal cancer. For instance, comparable concentrations of 2- and 4-OHE2 have been found in the kidney and liver of male Syrian hamsters treated with E2, despite the fact that renal activities of estrogen-2- and 4-hydroxylases are only one-tenth to one-twentieth the hepatic values (97). These comparable estrogen concentrations suggest that, in addition to direct aromatic hydroxylation of parent hormones, other metabolic pathways of CE formation exist in the kidney, such as deconjugation of methoxyestrogens and glucuronide metabolites. In extrahepatic tissue, such as in the hamster kidney, deconjugation reactions may be as important a source of CEs as aromatic hydroxylation of parent hormone.
Metabolic Activation by Redox Cycling
CEs, including 4-OHE2, are capable of metabolic redox cycling. This process consists of the organic hydroperoxide-dependent oxidation of the CEs (the hydroquinone) to the quinone and the NADPH-dependent CYP reductase-catalyzed reduction of the quinone intermediate back to the hydroquinone (Fig. 8) (68). The quinones may react with nucleic acids and form stable or unstable DNA adducts, as described in Chapter 4. The semiquinone free radical is an intermediate in each of these conversions. The estrogen semiquinone may react with molecular oxygen and form quinone and superoxide radicals (98). Thus, metabolic redox cycling is a mechanism of metabolic activation resulting in the continuous formation of free radical species from possibly small amounts of CE substrate (Fig. 8).
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Superoxide radicals may be reduced enzymatically or nonenzymatically to hydrogen peroxide and further to hydroxy radicals, which are capable of initiating lipid peroxidation. Lipid hydroperoxides are, thus, formed by metabolic redox cycling of CEs and, in turn, support the formation of CEs and their redox cycling. This is achieved by lipid hydroperoxides functioning as cofactors for the organic hydroperoxide-dependent estrogen-2/4-hydroxylase (78,86) and for CYP1A1, which catalyzes the oxidation of hydroquinones, including CEs, to corresponding quinones (99). This shift toward lipid hydroperoxides as cofactors for enzyme-catalyzed reactions represents a loss of metabolic control in the cell, which is exerted in part by the bioavailability of NADPH as cofactor for biosynthetic reactions. Therefore, metabolic redox cycling of CEs, including 4-OHE2, may persist indefinitely in an unregulated fashion. Moreover, this redox cycling process amplifies damage because relatively small amounts of CEs may repeatedly undergo this process and generate greater than stoichiometric amounts of various radical species.
Conclusions
4-OHE2 is a carcinogenic metabolite and may be formed by four different processes. 4-Hydroxyestrogens may be generated: (a) as a minor byproduct of NADPH-dependent 2-hydroxylation of either E1 or E2 due to a lack of specificity of the estrogen-2-hydroxylases; (b) by specific NADPH-dependent 4-hydroxylation of E1 or E2 catalyzed by CYP1B1 and possibly other 4-hydroxylases; (c) by organic hydroperoxide-dependent estrogen-2- and 4-hydroxylase activity, which forms these two catechols in approximately equal amounts [little is known about the location and regulation of this enzyme(s)]; and (d) by deconjugation of 4-methoxyestrogens and other CE conjugates. Tumors are expected to arise in cells or tissues that experience high concentrations of 4-OHE2 or 4-OHE1 formed by one or several of the processes cited above. High concentrations of 4-hydroxyestrogens may be found in cells with high formation of CEs by aromatic hydroxylation of parent estrogens, inadequate phase II metabolism of these catechols, or high rates of deconjugation of phase II metabolites back to CEs. Thus, CE metabolite concentrations are the result of a balance of several processes of formation and catabolism and may be unique for a given cell type, depending on its specific profile of metabolizing and catabolizing enzyme activities.
Elevated concentrations of 4-hydroxylated estrogens may be formed as a result of a loss of regulatory control of cells because organic hydroperoxides may be cofactors for the formation and metabolic redox cycling of CEs. In other cell types, 4-OHE2 may be formed by specific 4-hydroxylation of E2. The unique distribution of estrogen-4-hydroxylases in tissues, such as the uterus, breast, and others, points to 4-OHE2 as a hormone with characteristics distinct and different from those of E2. Specific 4-OHE2-induced processes, such as blastocyst implantation in the uterus of mice (100), support this view. Therefore, the organ-specific distribution of E2–4-hydroxylases may be a function of the role of this catechol in physiologic processes.
Tumors may be formed in organs and cells, which experience a loss of regulatory control as metabolism is converted to an organic hydroperoxide-dependent process from an NADPH-dependent process. Tumors may also arise in cells that express estrogen-4-hydroxylase activity and limited phase II metabolism, as may occur in mammary or uterine cells. More research is needed to examine the metabolic characteristics of each cell type in these organs and, thus, their potential for high CE concentrations and for elevated tumor risk.
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ESTROGEN 4-HYDROXYLATION CATALYZED BY HUMAN CYTOCHROME P4501B1: OXIDATIVE METABOLISM OF ESTROGENS |
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TopNotesAbstractIntroductionPotential Role of Aromatase...Metabolic Activation of...Estrogen 4-Hydroxylation...Identification and...Estrogen Hydroxylation Catalyzed...References |
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The oxidative metabolism of estrogens in vivo, especially E2 and E1, is known to occur at several positions, including carbons C-1, C-2, C-4, C-6, C-7, C-11, C-14, C-15, C-16, and C-18 [reviewed in (52,67)]. Studies of the routes of formation of these estrogen metabolites comprise an active area of research in several scientific fields, including endocrinology, pharmacology, environmental health sciences, oncology, and epidemiology. Additional knowledge of the rates of formation, metabolic fate (including further metabolism and routes of elimination), and activities of these hydroxylated steroids will support the scientific assessment of the relative importance of such metabolic pathways to hormonal carcinogenesis and other hormone-related diseases.
The mechanism of carcinogenesis of estrogens has been primarily attributed to their specific action as steroid hormone receptor agonists, controlling cellular growth and differentiation in estrogen-responsive tissues through concerted gene regulation. As discussed more fully in other sections of this monograph (see Chapters 3 and 4), increasing evidence of an additional mechanism of carcinogenesis has focused attention on the CE metabolites, which are less potent estrogens than E2 yet are biologically reactive, capable of directly or indirectly damaging protein, lipid, and DNA (101–103). Worthy of reiteration, our present knowledge of the mechanisms of estrogen-related disease processes indicates a strong estrogen receptor-mediated action. While the activities and potencies of the various estrogen metabolites are less understood, it is reasonable to predict, and in certain cases known, that the activities of these metabolites range from inactive to toxic to protective. Regarding estrogen-related cancers, the routes and rates of formation of certain estrogen metabolites, specifically the 2-, 16-, and more recently the 4-hydroxyestrogens, are being investigated as etiologic factors. Of particular interest to this research focus group (The Cancer Cube) and a major topic of the conference preceding this monograph are the local production and activity of 4-OHE2.
4-Hydroxylated metabolites represent only a small percentage of the total amount of estrogens detected in the urine, and 4-hydroxylation was previously considered a minor metabolic route (104). However, tissue 4-hydroxylation of E2 may play an important role in estrogen homeostasis. In human (87) and mouse (80) uteri, rat pituitary (79), and hamster kidney (105), the rate of E2-4-hydroxylation equals or exceeds the rate of 2-hydroxylation, and, notably, these organs are sites of estrogen-induced tumors (62,81,106,107). In comparison to normal tissue, elevated E2-4-hydroxylase activity has been observed in human tissue samples prepared from breast (88,108) and uterine (87) tumors. Furthermore, in male hamster kidney, the carcinogenic and DNA-damaging activity of 4-OHE2 and the lack of activity of 2-OHE2 (83,85,109) implicate the 4-hydroxylated metabolites in estrogen carcinogenesis (11). Evidence of the toxicity of CEs via the generation of free radicals through lipid hydroperoxide-supported (110) reductive-oxidative cycling mechanisms continues to accumulate (111–113). Furthermore, several laboratories (71,114,115) have demonstrated that estrogen-3,4-quinones can form DNA adducts, indicating the direct genotoxic potential of these compounds. Requisite to elucidating the contributions of the 4-hydroxyestrogens to estrogen carcinogenicity are the identification and characterization of the enzymes that produce these metabolites.
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IDENTIFICATION AND CHARACTERIZATION OF HUMAN CYP1B1 |
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TopNotesAbstractIntroductionPotential Role of Aromatase...Metabolic Activation of...Estrogen 4-Hydroxylation...Identification and...Estrogen Hydroxylation Catalyzed...References |
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A novel human cytochrome P450 was first isolated by differential hybridization as a TCDD-responsive complementary DNA (cDNA) clone from a human keratinocyte cell line treated with TCDD. Levels of this messenger RNA (mRNA) (P4501B1) were shown to be increased 50-fold by treatment with 10 nM TCDD, in part as the result of increased rates of gene transcription (116). Analysis of the complete cDNA sequence of this 5.1-kilobase (kb) TCDD-inducible mRNA identified a new gene subfamily of cytochrome P450, CYP1B1, based on 40% sequence homology to other polycyclic aromatic hydrocarbon (PAH)-inducible isoforms, CYP1A1 and CYP1A2 (90). The CYP1B1 gene was mapped to human chromosome 2 by polymerase chain reaction (PCR) amplification of human/rodent somatic cell hybrid panels using specific primers to the 3`-untranslated region of the CYP1B1 cDNA. Segregation analysis of the data showed 100% concordance between the presence of the PCR product specific for CYP1B1 and chromosome 2 and greater than 8% discordance for all other chromosomes. Southern blot analysis of human genomic DNA, using a single cDNA probe corresponding to the 5`-portion of the CYP1B1 open reading frame, suggested the presence of only a single gene in this subfamily (90).
In the 1980s through studies on the metabolism of PAH, Jefcoate and co-workers (117) found that several rodent tissues, including embryo fibroblasts, adrenals, and the mammary gland, produced anomalous product ratios suggesting the presence of a novel P450 cytochrome. This was established 10 years later by the purification of a novel P450 that was found in each of these tissues. Subsequently, Jefcoate and co-workers tried to address the function of this form, notably by characterizing its expression pattern and regulation. They used antibodies raised against the purified protein to identify expression of equivalent human forms in human breast cells and keratinocytes (118), as well as in a variety of rodent steroidogenic cells (adrenal, testis, ovary) (119). Expression in drug-metabolizing organs, like the liver, was low. This suggested that the function was physiologic in these nonhepatic tissues (i.e., the mammary gland). This was supported by the strong hormonal regulation demonstrated in steroidogenic cells. The induction of this P450 cytochrome by PAH was established, suggesting involvement of the aryl hydrocarbon (Ah) receptor. Cloning of the mouse and rat cDNAs from mouse embryo fibroblasts and rat adrenals, respectively (91,120), completed the initial characterization of this P450. This characterization allowed Jefcoate's laboratory to confirm that this rodent P450 had the same characteristics of and high sequence homology (82%) to human CYP1B1.
In human tissues, Northern blot analysis of CYP1B1 expression showed that CYP1B1 mRNA could be detected in each of 15 different tissue RNA samples prepared from heart, brain, placenta, lung, liver, skeletal muscle, kidney, spleen, thymus, prostate, testis, ovary, small intestine, colon, and peripheral blood leukocytes. In primary cultures of normal human epidermal keratinocytes treated for 24 hours with 10 nM TCDD, levels of CYP1B1 mRNA were increased more than 70-fold (90), demonstrating that TCDD induction of CYP1B1 expression in keratinocytes was not restricted to the transformed cell line SCC-12F (116).
Isolation and initial characterization of the human P4501B1 gene (121) described the DNA sequence of a 12-kb genomic clone corresponding to the entire 5.1-kb CYP1B1 cDNA (90) and containing 3.0 kb of upstream (5`- of the ATG) DNA (123). Comparison of these sequences revealed the presence and positions of three exons (371, 1044, and 3707 base pairs [bp]) and two introns (390 and 3032 bp), with the CYP1B1 open reading frame spanning exons 2 and 3. Southern blot analysis using cDNA probes corresponding to each of the three exons of CYP1B1 supported the presence of only a single CYP1B1 gene and excluded the existence of pseudogenes (121). High-resolution chromosome mapping confirmed the previous somatic cell hybrid analysis (90) and further mapped the CYP1B1 gene to human chromosome 2 at 2p21–22 (121). A single transcription initiation site was identified in this CYP1B1 gene, which lacks a consensus TATA box sequence. Deletion analysis of CYP1B1-promoter reporter-gene constructs identified a specific region (-1022 to -835) containing three TCDD-responsive enhancer-core-binding motifs (5`-GCGTG-3`) contributing to the TCDD-inducible expression of CYP1B1 in the human keratinocyte cell line SCC-12F (121).
Comparison of the human CYP1B1 genomic and cDNA sequences, obtained independently from two cell lines derived from different individuals, revealed three sequence differences (allelic variants, potential polymorphisms). Concurrent human genetic studies to identify one of two loci for primary congenital glaucoma (PCG) led to the mapping of the PCG locus GCL3A to human chromosome 2 at the 2p21 region [reviewed in (122)]. Subsequent investigations (123–125) have led to independent reports that identify distinct CYP1B1 gene mutations that segregate with the GCL3A phenotype in PCG families. GCL3A-linked PCG has an autosomal recessive mode of transmission, and it appears that the observed mutations will result in the absence of P4501B1 protein and/or activity. Nonetheless, further understanding of this disease will require additional knowledge of the physiologic function of CYP1B1.
As an added benefit of the extensive DNA sequence analysis of the translated regions of the CYP1B1 gene in 22 PCG families and in 100 randomly selected normal individuals (124), the Val 432–Leu CYP1B1 polymorphism (90,121) was confirmed. Three additional polymorphisms predicting variant amino acid sequences were identified: Arg 48–Gly, Ala 119–Ser, and Asn 453–Ser. The frequency of each wild-type allele, calculated for the reported sample of 100 normal individuals (124), is as follows: Arg 48, 0.71; Ala 119, 0.71; Val 432, 0.28; and Asn 453, 0.76. The identification of several frequently occurring polymorphisms in the human CYP1B1 gene demonstrates the strength of direct genomic sequence analysis as a method to identify single nucleotide polymorphisms. At this time, the appropriate procedures to determine the functional significance of such variant proteins are less clear, making this an active area of research. Nonetheless, epidemiologic studies, similar to those reported for catechol-O-methyltransferase polymorphisms and the risk for breast cancer (126,127), are under way for CYP1B1. Such studies should further clarify the role of CYP1B1, specifically, and hydroxylated estrogen metabolites, generally, in estrogen-related diseases.
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ESTROGEN HYDROXYLATION CATALYZED BY HUMAN P4501B1 |
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TopNotesAbstractIntroductionPotential Role of Aromatase...Metabolic Activation of...Estrogen 4-Hydroxylation...Identification and...Estrogen Hydroxylation Catalyzed...References |
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The widespread clinical use of antiestrogens in the adjuvant treatment of breast cancer provided a strong rationale for studies to determine the mechanism(s) of the antiestrogenic activity of ligands for the Ah receptor (128,129). In the MCF-7 breast tumor cell line, a sensitive gas chromatography/mass spectrometry method of analysis was used to show that treatment with TCDD resulted in large, more than 10-fold, increases in the rates of hydroxylation of E2 at positions C-2, C-4, C-15
, and C-6 (129). These studies support the role of increased E2 metabolism in the observed antiestrogenic effects of TCDD, which include the inhibition of estrogen-mediated expression of tissue plasminogen-activator activity and the formation of multicellular foci (130,131). Spink et al. (130) convincingly demonstrated that P4501A1 catalyzed the hydroxylation of E2 at the C-2, C-15, and C-6 positions in MCF-7 cells treated with TCDD. However, the metabolism at the C-4 position was shown to be distinct, catalyzed by an unknown TCDD-inducible CYP, best characterized as a low-Km E2-4-hydroxylase. Isolation and characterization of the TCDD-inducible CYP1B1 from humans (90,116) and from rodent species (91,120,131) and the observation that human CYP1B1 mRNA was elevated by treatment of human breast cell lines with TCDD led to studies of CYP1B1 expression and activity in MCF-7 cells (89). Antibodies raised against mouse CYP1B1 (anti-P450-EF) and cross-reactive with human CYP1B1 (117,118) were used to investigate E2 hydroxylation catalyzed by microsomes prepared from TCDD-treated MCF-7 cells. This antibody preparation was shown to selectively inhibit E2-4-hydroxylation in a concentration-dependent manner. Furthermore, the elevated expression of P4501B1 protein and mRNA in these TCDD-treated cells closely paralleled the observed rates of E2 4-hydroxylation (89). Collectively, these results provided strong support for the assignment of human CYP1B1 as the low-Km E2-4-hydroxylase that is induced in MCF-7 human breast cancer cells by TCDD treatment.
To establish the specific relationship between the P4501B1 gene product and E2 metabolism, human P450 protein was expressed in Saccharomyces cerevisiae (3). Two recombinant P4501B1 expression plasmids were described. One construct, identified as CYP1B1
0, was designed to express a protein of 543 amino acids, corresponding to the entire deduced amino acid sequence of P4501B1. A second expression construct, identified as CYP1B13, was designed to produce a protein that did not contain the three amino acid residues after the initial methionine (deletion of amino acids 2–4). The design of the second plasmid was based on the reported amino acid sequence of rat P4501B1, showing that this protein did not contain the first four amino acid residues of its corresponding amino acid sequence (120). Levels of P4501B1 protein present in the microsomal fraction of the CYP1B13 construct (340 pmol/mg protein) were about 10-fold greater than the levels of P450 of CYP1B10 preparations (3). This difference in expression of P450 may be important, since a similar construct containing this same deletion of amino acids 2–4 also resulted in the highest expression levels of human P4501B1 in Escherichia coli (132). Further studies of the translation, processing, and subcellular localization of this protein are warranted. Yeast CYP1B10 microsomal preparations, containing the lower P4501B1-specific content, were determined to have the greatest E2 hydroxylation turnover numbers; these rates were not increased by the addition of NADPH cytochrome P450 reductase to the reaction mixture. Reactions containing these microsomes were shown to catalyze the 4- and 2-hydroxylation of E2, with Km values of 0.71 and 0.78 µM and turnover numbers of 1.39 and 0.27 nmol product/minute per nmol P450, respectively (3).
The major CEs detected in serum and urine are the 2-hydroxylated metabolites. The liver is the primary site of estrogen metabolism, where rates of 2-hydroxylation, catalyzed by P4501A2, 3A3, and 3A4, greatly exceed the rate of 4-hydroxylation. The reported apparent Km values for E2 hydroxylation catalyzed by the human forms of these enzymes range from 20 to 156 µM, which are considerably higher that the apparent Km values of P4501B1, less than 1 µM (Table 4) (3,132–137). The turnover number for the formation of 4-OHE2 by P4501B1 is similar to the turnover numbers for the formation of 2-OHE2 by human P4501A2, 3A3, and 3A4. The E2-4-hydroxylase activity of P4501B1 has the highest catalytic efficiency (turnover/Km) of any reported estrogen hydroxylase, and the apparent Km values of E2-4- and 2-hydroxylase activities are the lowest reported values for estrogen hydroxylation (Table 4) (3). Comparisons of the values presented in Table 4 indicate a potential role of P4501B1 hydroxylase activity in estrogen homeostasis, especially in extrahepatic organs, which express much lower levels of P4501A2 and 3A4 than does the liver (138).
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In further experiments to explore the significance of the low-Km 4-hydroxylase activity of P4501B1, cellular E2 metabolism was studied in MCF-7 cells treated with the potent Ah receptor ligand, indolo[3,2b]carbazole (ICZ) (139). MCF-7 cells exhibited ICZ concentration-dependent increases in P4501B1 and 1A1 mRNA levels. In parallel experiments to determine E2 metabolism, treatment of MCF-7 cells with 10 µM ICZ for 72 hours, followed by replacement of medium containing 1 µM E2 for 6 hours, resulted in a TCDD-like profile of E2 metabolites (129), with increased rates of hydroxylation occurring at the C-2, C-4, C-6
, and C-15 positions of E2; the rates of 2-hydroxylation were the greatest, approximately threefold to sevenfold greater than the rates of hydroxylation at the other positions. When the E2 concentration was decreased to 10 nM, the rates of 2- and 4-hydroxylation in ICZ-treated cells were detectable and approximately equal, demonstrating that the E2 4- and 2-hydroxylation activities of P4501B1 are significant at low, physiologically relevant concentrations of E2 (3). In summary, these studies demonstrate that human P4501B1 is a catalytically efficient E2-4-hydroxylase that is likely to participate in endocrine regulation and estrogen-related disease. Tissue Expression of Human P4501B1
Large differences are demonstrated between species in CYP1B1 metabolism of PAH (5,140). Thus, the question arose whether the expression of CYP1B1 in breast cells gives any indication of physiologic or pathologic functions linked to such activity. The relative expression levels of CYP1A1 and CYP1B1 will determine the conversion of E2 to the 2- and 4-hydroxylation products, respectively. CYP1B1 is expressed constitutively in cultured breast luminal epithelial cells, which are the source of most breast tumors (5). This implies that there will be a low basal formation of 4-CE in all tumors. Typically, CYP1A1 is essentially undetectable in breast tissue from most donors. Environmental activators of the Ah receptor, such as polychlorobiphenyls, which have been detected at considerable levels in breast fat, could potentially induce both forms. This certainly is the case for these chemicals in cultured breast epithelial cells (5). However, analysis of CYP1A1 mRNA in human breast tissue by reverse transcription–PCR (RT–PCR) indicated that this isoform is typically undetectable. Low levels are occasionally present in cultured epithelial cells, but this is retained independent of medium changes and is almost certainly constitutive. These data indicate that relatively little 2-CE will be produced in vivo. A central question to be addressed and resolved in future research is whether this very low formation of 4-CE is physiologically or pathologically significant. The relatively high affinity of CYP1B1 for E2 allows this activity to be retained even at low physiologic hormone levels. While it might seem that this activity is too low to remove E2, a substantial proportion of CYP1B1 is localized in nuclear and perinuclear membranes. This may exert a relatively greater effect on nuclear levels of E2 and 4-CE production. This constriction to depletion of estrogen will depend on the relative activity of other metabolic processes acting on E2. CYP1B1 seems to be the major source of 4-CE formation, and the measurement of this steroid in vivo points to substantial accumulated activity.
Information on the "basal" and inducible expression of P4501B1 in human cells and tissues continues to be an interesting, yet less developed, aspect of the knowledge of this recently identified CYP. Additional knowledge of tissue P4501B1-specific content and activity will strengthen scientific assessments of the relative importance of estrogen metabolic pathways to estrogen-related diseases and their prevention.
As stated above, the initial characterization of human P4501B1 revealed that levels of this 5.1-kb transcript were detectable in multiple adult tissue samples, the kidney sample exhibiting the greatest apparent signal relative to the other samples tested (90). In a subsequent study (4), these results were extended to include analyses of additional adult tissue samples representing five additional organs and a second kidney sample, comparative analyses of the relative expression of P4501A1 and 1A2 in these same tissue samples, and analyses of tissue samples from five fetal organs. P4501A1 mRNA was detected in 12 of the 21 adult tissue RNA samples but in none of the five fetal tissue RNA samples. The most intense hybridization signals occurred in the prostate and mammary tissues. P4501A2 mRNA was detected only in the adult liver sample. P4501B1 mRNA was detected in 20 of the 21 adult RNA samples (heart, brain, placenta, lung, liver, skeletal muscle, kidney 1, kidney 2, spleen, thymus, prostate, testis, ovary, small intestine, colon, peripheral blood leukocytes, adrenal, pituitary, uterus, and mammary tissue) and in fetal heart, brain, lung, and kidney. The levels of P4501B1 RNA in adult kidney of both sexes and fetal kidney, as well as in prostate, uterus, and mammary tissue, were apparently greater than those in other tissues (4). Many of these findings were confirmed in a report describing an RT–PCR-based analysis of P4501B1 expression showing that human P4501B1 is expressed mainly in extrahepatic tissues of adults and fetuses (141). Contrary to the earlier report (4), P4501B1 RNA was not detected in samples of adult lung (141). While the basis of this discrepancy is unlikely to be identified, other investigators (142) have reported the detectable expression of this mRNA in lung tissue samples from smokers. In summary, these preliminary analyses of the organ-specific RNA distribution of the P4501 gene family have revealed that, while P4501A2 is expressed primarily in the liver, P4501A1 and 1B1 are expressed widely and found in many of the same tissues. Furthermore, P4501B1 appears to be the predominant family 1 P450 expressed in human fetal tissues (4,141).
Regarding the expression of P4501B1 in organs such as breast and uterus, where strong associations between estrogen exposure and the risk for cancer are known, accumulating evidence indicates the presence and activity of this enzyme in these tissues. Elevated CE production has been associated with tumors of the breast (88,108,143,145), and P4501B1 RNA (4,145,146) and protein (147) have been detected in both normal (4,146) and tumor (145–147) breast tissue samples. Furthermore, in human uterine tissue, where 4-hydroxylation of E2 is increased in myomas compared with surrounding myometrium, the 4-hydroxylase activity of myoma microsomes was strongly inhibited by an antimouse P4501B1 antibody, suggesting that, in human uterine tissue, E2 4-hydroxylation is catalyzed by P4501B1 (87). In addition, Larsen et al. (5) demonstrated the expression of P4501B1 protein and activity in early-passage human mammary epithelial cells isolated from reduction mammoplasty tissue of seven individual donors. Specific contents of CYP1B1 and CYP1A1 protein in microsomal preparations (day 6 of culture) were quantitated by immunoblot analysis. Levels of constitutive CYP1B1 protein ranged from 0.01 to 1.4 pmol/mg microsomal protein; exposure to TCDD increased these levels, ranging from 2.3 to 16.6 pmol/mg microsomal protein. Levels of constitutive CYP1A1 protein were much lower than those of CYP1B1. However, the inductive response of CYP1A1 to TCDD treatment was very strong, resulting in comparable specific contents of the two P450 cytochromes in microsomes prepared from treated cells. This study indicates that human mammary epithelia constitutively expresses variable levels of functional CYP1B1 protein (11), which may contribute to the oxidative metabolism of E2 and other estrogens.
To aid in the analysis of CYP1B1 protein expression, our laboratory has produced polyclonal rabbit anti-P4501B1 antibodies that were shown to be both sensitive and CYP1B1 specific, detecting this protein by both immunoblot and immunohistochemical analyses (147,148). Recently, we (149) have used these antibodies to investigate the constitutive expression and cellular localization of P4501B1 in normal human tissue samples. A representative immunohistochemical analysis of P4501B1 protein expression in normal human breast tissue is presented in Fig. 9 (Sutter TR, Kim J, Sherman M: manuscript in preparation). As best seen in Fig. 9, panels B and C, P4501B1 is expressed ubiquitously in the ducts of this breast lobule, conspicuously present in the epithelia, as well as the myoepithelia, stromal fibroblast, and endothelial cells. P4501B1 staining is predominantly cytoplasmic, yet some nuclear staining is evident. Like the previous study (5), these results are consistent with the concept that human breast epithelial cells constitutively express significant amounts of CYP1B1 protein, which may contribute to the oxidative metabolism of E2 and other estrogens in this tissue.
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Conclusions
The kinetic parameters determined for the E2-4-hydroxylase activity of human P4501B1 establish this enzyme as the most catalytically efficient estrogen-hydroxylase described to date. This observation is important because it suggests that this enzyme is responsible for the E2-4-hydroxylase activity that has been observed in several tissues, such as human uterus and breast. The specific expression of P4501B1 and formation of CEs have been independently associated with estrogen-related tumors in multiple tissues and species. Further elucidation of the physiologic and/or pathologic significance of the E2-4- and 2-hydroxylase activities of P4501B1 will require additional knowledge of the tissue, cellular, and developmental expression (and regulation thereof) of this gene, and its protein product in humans and other animals. Despite the present knowledge gaps, our understanding of human P4501B1 as a low-Km E2-4- and 2-hydroxylase and a major extrahepatic form of CYP firmly supports the hypothesis of the role of CE metabolites in estrogen-related carcinogenesis.
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NOTES |
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R. J. Santen is on the advisory board of Lilly Pharmaceuticals, Indianapolis, IN.
Supported by Public Health Service (PHS) grants CA63129 and CA74971 (to J. G. Liehr) from the National Cancer Institute, National Institutes of Health (NIH), Department of Health and Human Services, and by PHS grants ES08148, ES03819, and ES07141 (to T. R. Sutter) from the National Institute of Environmental Health Sciences, NIH.
J. G. Liehr acknowledges the contributions of M. J. Ricci and Drs. B. T. Zhu, X. Han, D. Roy, A. Gladek, M. Y. Wang, and H. Bhat who contributed to these studies while at the University of Texas Medical Branch, Galveston. T. R. Sutter acknowledges and thanks all of the students, postdoctoral fellows, collaborators, colleagues, and friends who have contributed to the research described in this review; especially the contributions of Dr. William Greenlee (Chemical Industry Institute of Toxicology, Research Triangle Park, NC), Dr. David Spink (Wadsworth Center, Albany, NY), and Dr. Carrie Hayes (University of Memphis, TN). He thanks M. Sherman and J. Kim (The Johns Hopkins University, Baltimore, MD) for their contributions to Fig. 9
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