Accepted Manuscript Review Estrogen metabolites and breast cancer Richard J. Santen, Wei Yue, Ji-Ping Wang PII: DOI: Reference:
S0039-128X(14)00196-2 http://dx.doi.org/10.1016/j.steroids.2014.08.003 STE 7625
To appear in:
Steroids
Please cite this article as: Santen, R.J., Yue, W., Wang, J-P., Estrogen metabolites and breast cancer, Steroids (2014), doi: http://dx.doi.org/10.1016/j.steroids.2014.08.003
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65
Estrogen Metabolites and Breast Cancer
Richard J. Santen,1 Wei Yue1, and Ji-Ping Wang1, 1
Division of Endocrinology and Metabolism, Department of Internal Medicine, University of Virginia
Health Sciences System, PO box 801416, Aurbach Medical Research Building, Charlottesville,Virginia, 22908-1416
Corresponding author. Richard J.Santen MD Tel.: +1 434 924 2961/2207; fax: +1 434 924 1284.
E-mail address: [email protected] (R.J. Santen).
Key words: estrogen, metabolites, genotoxic, breast cancer, aromatase, adducts
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65
Highlights
x
Genotoxic metabolites of estradiol contribute to breast cancer development
x
Both estrogen receptor-mediated and estrogen receptor-independent effects of estradiol contribute to breast cancer development.
x
Aromatase inhibitors should be a more effective means of preventing breast cancer based on the proof of principle experiments described.
x
MCF-7breast cancer cells, aromatase transfected mouse breast, estrogen receptor knock out /Wnt 1 transgenic mouse breast and human breast tissue can convert estradiol to genotoxic metabolites
Abstract 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65
Epidemiologic studies link several factors related to estrogen production in women to an increased risk of breast cancer. These include early menarche, late menopause, obesity, use of post-menopausal hormone therapy, and plasma estradiol levels. Two possible mechanisms have been proposed to explain the increased risk: (1) estrogen receptor (ER) mediated stimulation of breast cell proliferation with a concomitant enhanced rate of mutations and (2) metabolism of estradiol to genotoxic metabolites with a resulting increase in DNA mutations. The metabolism of estradiol can cause DNA damage in two ways: (a) formation of estradiol-adenine -guanine adducts which are released from the DNA backbone leaving depurinated sites which undergo error prone DNA repair and mutations and (b) generation of oxygen free radicals resulting from redox cycling of 4-OH estradiol to the 3-4 estradiol quinone and back conversion to 4-OH estradiol. If one or both pathways are operative, VXI¿FLHQW QXPEHUV RI PXWDWLRQV DFFXPXODWH over a long period of time to induce neoplastic transformation. Our studies are based on the hypothesis that both receptor-mediated and genotoxic pathways contribute to breast cancer. We initially demonstrated that MCF-7 breast cancer cells and normal breast tissue in aromatase transfected mice contain the enzymes necessary to convert estradiol to the estradiol DNA adducts. We then utilized a highly reductionist model to separately analyze the effect of estrogen receptor alpha (ER) on tumor formation and the effects of estrogen depletion by castration in ER knock out /Wnt-1 (ERKO/Wnt) transgenic animals to assess the effects of estradiol in the absence of an ER. Estradiol was added back in castrate ERKO/Wnt animals to determine if .RFK¶V postulates could be fulfilled to increase the incidence of cancer with administration of exogenous estradiol. Finally, we assessed the effects of an aromatase inhibitor on tumor incidence in non-castrate, ERKO/Wnt animals. The studies demonstrated the conversion of estadiol to genotoxic metabolites in breast tissue. In addition, knockout of ERĮ caused a reduction in incidence of tumor formation and a delay in the occurrence of those that formed. Oophorectomy further reduced the incidence of tumors and delayed their onset whereas estradiol add-back returned the incidence rate to that observed before oophorectomy. The aromatase inhibitor, letrozole, delayed the onset of tumor formation. Taken together, these data support a role for estradiol metabolism as one of the components in the development of experimental breast cancer.
Author's personal copy Introduction: 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65
Several clinical and experimental observations suggest a mechanistic link between estradiol production in women and the development of breast cancer. A compelling observation is that breast cancer occurs 100 fold more frequently in women than in men and mean E2 levels are an order of magnitude higher in women than in men (1).Bilateral oophorectomy before age 35 reduces the lifetime incidence of breast cancer by 75% (2). Increased lifetime exposure to estrogen as inferred by early menarche, late menopause, high bone density and obesity is associated with an increased risk of breast cancer (1). Data from two separate studies demonstrated an increasing risk of breast cancer as a function of plasma free-E2 levels (3;4). In these studies, women in the highest quintile of plasma E2 experienced a 2.5-fold higher rate of breast cancer over the years than those in the lowest quintile. Blockade of estrogen action with tamoxifen or raloxifene reduces the incidence of breast cancer by 38% (5-7) and with aromatase inhibitors by 51-65% in high risk women. Finally, inhibition of E2 synthesis with aromatase inhibitors or abrogation of its action with anti-estrogens prevents the development of contralateral breast cancer during adjuvant therapy (8). Experimental data in animals also support a role for estrogens in the development of breast neoplasms. E2 administration causes breast cancer in various animal models and anti-estrogens abrogate this effect (9). Taken together, these data provide substantial evidence for a role of estrogens in breast cancer development. The mechanisms whereby estrogens enhance the incidence of breast cancer are incompletely understood and the subject of some controversy. Estrogens could cause de novo breast cancer though either receptor-dependent or -independent mechanisms (Figure 1). Through actions mediated by its receptor, E2 enhances cell proliferation, a factor causally related to breast cancer development (10). The chances for errors in DNA replication and resulting mutations increase pari passu as the number of cell divisions increases. Estrogens and to a greater extent progestogens in conjunction with estrogens increase the rate of proliferation of immature and mature human breast tissue (11). Cell proliferation is greatest in the adult breast when E2 and progesterone levels are at their highest. Hofseth and colleagues have shown that menopausal hormone therapy with either estrogen or estrogen plus a progestogen increase proliferation in terminal duct lobular units, the putative site of breast cancer development t(11). This linkage of proliferation rate to the carcinogenic process represents the conceptual underpinning of the role of ER dependent mechanisms in breast carcinogenesis. The ER independent, carcinogenic effects of estrogens are believed to occur through the actions of estrogen metabolites (Figure 1 )(12;13). Because of the presence of the aromatic Aring, oxidative metabolism of E2/E1 results in the formation of 2,3- and 3,4-catechols catalyzed by phase I cytochrome P450 enzymes such as 3A4, 1A1, and 1B1 present in human breast tissue. CYP1B1 predominately catalyzes formation of the 4-OH catechol and the others, predominately the 2-OH catechol. In particular, the 3,4-quinones form unstable depurinating adducts with adenine and guanine, 4-OH-E2/E1-1-N3Adenine and 4-OH-E2/E1-1-N7Guanine as shown in Figure 2. These adducts undergo spontaneous depurination from the DNA backbone causing the formation of apurinic sites that undergo error prone DNA repair with resultant point mutations. The 2,3-quinones, on the other hand, form relatively stable DNA adducts and depurination occurs uncommonly (14). The 2,3- and 3,4-quinones can also be reduced to semiquinones by cytochrome P450 reductase, and this process can establish a redox cycle to produce reactive oxygen species that cause oxidative DNA damage (e.g., 8-oxo-deoxyguanine) (15). Experimental data suggest that the 4-OH metabolites may be more carcinogenic than their 2-OH counterparts (12;16) . We postulated that distinction between receptor-dependent and -independent actions could be best elucidated by experimental animal models using knock-out technology. Bocchinfuso et al. utilized their ERKO/Wnt-1 model to directly demonstrate the role of ERa in the carcinogenesis process (17). Our studies provided proof of the principle that both ER-dependent and ERindependent actions contribute to the carcinogenic process.
Author's personal copy Material and Methods 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65
Animals: ERKO/Wnt-1 and Wnt-(5í KHWHUR]\JRXV PLFH ZHUH REWDLQHG IURP WKH 1ational Institute of Environmental Health Sciences, Research Triangle Park (17). Wnt-1 transgenic animals were cross- bred with heterozygous ERa knockout mice to generate the Wnt- 1 dual transgenic mice, which could then be further bred to produce ERa knockout/Wnt-1 double transgenics (ERKO/Wnt-1). Full characterization of the phenotypic, biologic and biochemical properties of these animals have been published previously(17;17). µµ(FODPS¶¶PHWKRGDQGGUXJDGPLQLVWUDWLRQ: Silastic tubes of 0.19 cm internal diameter were ¿OOHG ZLWK (FKROHVWHURO PL[WXUHV DW YDULRXV UDWLRV Our prior studies validated the ability to µµFODPS¶¶SODVPD(DWOHYHOVUDQJLQJIURPWRSJPORYHUD- month period (18). In our study, we clamped estradiol at early follicular phase (80 pg/ml) and mid-luteal phase (240 pg/ml) levels in women Letrozole was suspended in 0.3% carboxymethyl cellulose solution in saline and administered by subcutaneous injection once a day at a dosage of 20 mg/ mouse, 5 days a week. Estrogen metabolites: Estradiol metabolites, conjugates and depurinating DNA adducts were measured by HPLC with a 12-channelelectrochemical detector (16;19;19) Confirmation with mass spectrometry was utilized to verify key measurements. These methods have been extensively described previously and will not be described further here Statistical methods: The Kaplan±Meier analyses were used to compare the tumor-free survival time between different treatment groups of mice in the study. The rate of tumor development was compared among the various treatment groups to determine whether they are statistically sLJQL¿FDQWO\ GLIIHUHQW 7KH 6WXGHQW¶V W-test was used to compare mean uterine weights between 2 JURXSVDQGDVLJQL¿FDQFHOHYHORIZDVFRQVLGHUHGWREHVWDWLVWLFDOO\VLJQL¿FDQW
Results: Measurement of metabolites in MCF-7 breast cancer cells: Our initial experiments examined whether the enzymes responsible for formation of estrogen metabolites and conjugates were present in human breast cancer cells, as imputed from detection of metabolites (20). MCF-7 cells were incubated with 10 µM 4-OH-estradiol for 24 h before collecting media for later measurement of the various metabolites and conjugates (Figures 3 A-D). As shown in Fig. 3B, we detected 4-methoxy-estradiol as well as the quinone conjugates and the depurinating 4-OHestradiol-1-N7-guanine adduct and its estrone analogue. We next determined whether these cells could aromatize a sufficient amount of testosterone to estradiol to result in formation of the depurinating species. As shown in Fig. 3C, we detected 131 pg/ml of estrogen (92 pg/ml of estradiol and 39 pg/ml of estrone in the media) indicating the production of estrogens from aromatization. The 4-OH-estradiol-1-N7-guanine adduct (and its estrone analogue) was also present at a total concentration of 0.92 pg/ml as were the glutathione, cysteine, and N-acetylcysteine conjugates of estradiol-3,4-quinone and its estrone analogues. Finally, the aromatase inhibitor letrozole was capable of inhibiting the formation of the estrogens from a total of 131 pg/ml of E1 and E2 (Fig. 3C) to 2.8 pg/ml (Fig. 3D) and their downstream metabolites to undetectable levels in most cases. Measurement of metabolites in ERKO/Wnt-1 mammary tissue: Formation of 4-OH-estrogen metabolites is favored over those of the 2-OH species and the catechol-O-methyl-transferase pathway appears to be relatively inactive. We detected 10.9 pmol/g of 4-OH-E2 and 4-OH-E1 in
Author's personal copy mammary tissue, as well as a total of 2.3 pmol/g of conjugated estrogen quinone (Figure 4). No 4methoxy-estrogen metabolites were present. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65
Measurement of metabolites in aromatase transfected mouse mammary gland: As shown in Figure 5, the untreated tissues contained approximately 3pMol/gm amounts of the 3,4 estradiol-adenine adduct. We utilized tetramethoxystilbene (TMS), an inhibitor of CYP 1B1 to inhibit the formation of 4-OH estradiol in these animals. This resulted in marked suppression of the depurinated adduct. Finally, use of the aromatase inhibitor letrozole (CGS) also blocked formation of this adduct.
(5Į-dependent DQG (5 Į-independent effects on tumor development: We postulated that distinction between receptor-dependent and -independent actions might best be elucidated by an experimental animal model using knock-out technology. As shown in Fig. 6, we showed that 50% of mammary tumors arose by 5 months in the ER+ animals versus 11 months in those without ERa (i.e. ERKO animals)(21). This demonstrated WKH PHFKDQLVWLF HIIHFW RI (5Į on the incidence and timing of development of breast tumors in this model. We reasoned that castration, by lowering endogenous E2 and therefore the downstream metabolites, would reduce tumor incidence from levels observed in intact ERKO/Wnt-1 animals. For these experiments, we compared intact and castrate ERKO/Wnt-1 animals. Castration delayed tumor onset (50% incidence time) from 12 to 23 months and reduced tumor incidence from 80% to 50% (p
S0039-128X(14)00196-2 http://dx.doi.org/10.1016/j.steroids.2014.08.003 STE 7625
To appear in:
Steroids
Please cite this article as: Santen, R.J., Yue, W., Wang, J-P., Estrogen metabolites and breast cancer, Steroids (2014), doi: http://dx.doi.org/10.1016/j.steroids.2014.08.003
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65
Estrogen Metabolites and Breast Cancer
Richard J. Santen,1 Wei Yue1, and Ji-Ping Wang1, 1
Division of Endocrinology and Metabolism, Department of Internal Medicine, University of Virginia
Health Sciences System, PO box 801416, Aurbach Medical Research Building, Charlottesville,Virginia, 22908-1416
Corresponding author. Richard J.Santen MD Tel.: +1 434 924 2961/2207; fax: +1 434 924 1284.
E-mail address: [email protected] (R.J. Santen).
Key words: estrogen, metabolites, genotoxic, breast cancer, aromatase, adducts
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65
Highlights
x
Genotoxic metabolites of estradiol contribute to breast cancer development
x
Both estrogen receptor-mediated and estrogen receptor-independent effects of estradiol contribute to breast cancer development.
x
Aromatase inhibitors should be a more effective means of preventing breast cancer based on the proof of principle experiments described.
x
MCF-7breast cancer cells, aromatase transfected mouse breast, estrogen receptor knock out /Wnt 1 transgenic mouse breast and human breast tissue can convert estradiol to genotoxic metabolites
Abstract 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65
Epidemiologic studies link several factors related to estrogen production in women to an increased risk of breast cancer. These include early menarche, late menopause, obesity, use of post-menopausal hormone therapy, and plasma estradiol levels. Two possible mechanisms have been proposed to explain the increased risk: (1) estrogen receptor (ER) mediated stimulation of breast cell proliferation with a concomitant enhanced rate of mutations and (2) metabolism of estradiol to genotoxic metabolites with a resulting increase in DNA mutations. The metabolism of estradiol can cause DNA damage in two ways: (a) formation of estradiol-adenine -guanine adducts which are released from the DNA backbone leaving depurinated sites which undergo error prone DNA repair and mutations and (b) generation of oxygen free radicals resulting from redox cycling of 4-OH estradiol to the 3-4 estradiol quinone and back conversion to 4-OH estradiol. If one or both pathways are operative, VXI¿FLHQW QXPEHUV RI PXWDWLRQV DFFXPXODWH over a long period of time to induce neoplastic transformation. Our studies are based on the hypothesis that both receptor-mediated and genotoxic pathways contribute to breast cancer. We initially demonstrated that MCF-7 breast cancer cells and normal breast tissue in aromatase transfected mice contain the enzymes necessary to convert estradiol to the estradiol DNA adducts. We then utilized a highly reductionist model to separately analyze the effect of estrogen receptor alpha (ER) on tumor formation and the effects of estrogen depletion by castration in ER knock out /Wnt-1 (ERKO/Wnt) transgenic animals to assess the effects of estradiol in the absence of an ER. Estradiol was added back in castrate ERKO/Wnt animals to determine if .RFK¶V postulates could be fulfilled to increase the incidence of cancer with administration of exogenous estradiol. Finally, we assessed the effects of an aromatase inhibitor on tumor incidence in non-castrate, ERKO/Wnt animals. The studies demonstrated the conversion of estadiol to genotoxic metabolites in breast tissue. In addition, knockout of ERĮ caused a reduction in incidence of tumor formation and a delay in the occurrence of those that formed. Oophorectomy further reduced the incidence of tumors and delayed their onset whereas estradiol add-back returned the incidence rate to that observed before oophorectomy. The aromatase inhibitor, letrozole, delayed the onset of tumor formation. Taken together, these data support a role for estradiol metabolism as one of the components in the development of experimental breast cancer.
Author's personal copy Introduction: 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65
Several clinical and experimental observations suggest a mechanistic link between estradiol production in women and the development of breast cancer. A compelling observation is that breast cancer occurs 100 fold more frequently in women than in men and mean E2 levels are an order of magnitude higher in women than in men (1).Bilateral oophorectomy before age 35 reduces the lifetime incidence of breast cancer by 75% (2). Increased lifetime exposure to estrogen as inferred by early menarche, late menopause, high bone density and obesity is associated with an increased risk of breast cancer (1). Data from two separate studies demonstrated an increasing risk of breast cancer as a function of plasma free-E2 levels (3;4). In these studies, women in the highest quintile of plasma E2 experienced a 2.5-fold higher rate of breast cancer over the years than those in the lowest quintile. Blockade of estrogen action with tamoxifen or raloxifene reduces the incidence of breast cancer by 38% (5-7) and with aromatase inhibitors by 51-65% in high risk women. Finally, inhibition of E2 synthesis with aromatase inhibitors or abrogation of its action with anti-estrogens prevents the development of contralateral breast cancer during adjuvant therapy (8). Experimental data in animals also support a role for estrogens in the development of breast neoplasms. E2 administration causes breast cancer in various animal models and anti-estrogens abrogate this effect (9). Taken together, these data provide substantial evidence for a role of estrogens in breast cancer development. The mechanisms whereby estrogens enhance the incidence of breast cancer are incompletely understood and the subject of some controversy. Estrogens could cause de novo breast cancer though either receptor-dependent or -independent mechanisms (Figure 1). Through actions mediated by its receptor, E2 enhances cell proliferation, a factor causally related to breast cancer development (10). The chances for errors in DNA replication and resulting mutations increase pari passu as the number of cell divisions increases. Estrogens and to a greater extent progestogens in conjunction with estrogens increase the rate of proliferation of immature and mature human breast tissue (11). Cell proliferation is greatest in the adult breast when E2 and progesterone levels are at their highest. Hofseth and colleagues have shown that menopausal hormone therapy with either estrogen or estrogen plus a progestogen increase proliferation in terminal duct lobular units, the putative site of breast cancer development t(11). This linkage of proliferation rate to the carcinogenic process represents the conceptual underpinning of the role of ER dependent mechanisms in breast carcinogenesis. The ER independent, carcinogenic effects of estrogens are believed to occur through the actions of estrogen metabolites (Figure 1 )(12;13). Because of the presence of the aromatic Aring, oxidative metabolism of E2/E1 results in the formation of 2,3- and 3,4-catechols catalyzed by phase I cytochrome P450 enzymes such as 3A4, 1A1, and 1B1 present in human breast tissue. CYP1B1 predominately catalyzes formation of the 4-OH catechol and the others, predominately the 2-OH catechol. In particular, the 3,4-quinones form unstable depurinating adducts with adenine and guanine, 4-OH-E2/E1-1-N3Adenine and 4-OH-E2/E1-1-N7Guanine as shown in Figure 2. These adducts undergo spontaneous depurination from the DNA backbone causing the formation of apurinic sites that undergo error prone DNA repair with resultant point mutations. The 2,3-quinones, on the other hand, form relatively stable DNA adducts and depurination occurs uncommonly (14). The 2,3- and 3,4-quinones can also be reduced to semiquinones by cytochrome P450 reductase, and this process can establish a redox cycle to produce reactive oxygen species that cause oxidative DNA damage (e.g., 8-oxo-deoxyguanine) (15). Experimental data suggest that the 4-OH metabolites may be more carcinogenic than their 2-OH counterparts (12;16) . We postulated that distinction between receptor-dependent and -independent actions could be best elucidated by experimental animal models using knock-out technology. Bocchinfuso et al. utilized their ERKO/Wnt-1 model to directly demonstrate the role of ERa in the carcinogenesis process (17). Our studies provided proof of the principle that both ER-dependent and ERindependent actions contribute to the carcinogenic process.
Author's personal copy Material and Methods 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65
Animals: ERKO/Wnt-1 and Wnt-(5í KHWHUR]\JRXV PLFH ZHUH REWDLQHG IURP WKH 1ational Institute of Environmental Health Sciences, Research Triangle Park (17). Wnt-1 transgenic animals were cross- bred with heterozygous ERa knockout mice to generate the Wnt- 1 dual transgenic mice, which could then be further bred to produce ERa knockout/Wnt-1 double transgenics (ERKO/Wnt-1). Full characterization of the phenotypic, biologic and biochemical properties of these animals have been published previously(17;17). µµ(FODPS¶¶PHWKRGDQGGUXJDGPLQLVWUDWLRQ: Silastic tubes of 0.19 cm internal diameter were ¿OOHG ZLWK (FKROHVWHURO PL[WXUHV DW YDULRXV UDWLRV Our prior studies validated the ability to µµFODPS¶¶SODVPD(DWOHYHOVUDQJLQJIURPWRSJPORYHUD- month period (18). In our study, we clamped estradiol at early follicular phase (80 pg/ml) and mid-luteal phase (240 pg/ml) levels in women Letrozole was suspended in 0.3% carboxymethyl cellulose solution in saline and administered by subcutaneous injection once a day at a dosage of 20 mg/ mouse, 5 days a week. Estrogen metabolites: Estradiol metabolites, conjugates and depurinating DNA adducts were measured by HPLC with a 12-channelelectrochemical detector (16;19;19) Confirmation with mass spectrometry was utilized to verify key measurements. These methods have been extensively described previously and will not be described further here Statistical methods: The Kaplan±Meier analyses were used to compare the tumor-free survival time between different treatment groups of mice in the study. The rate of tumor development was compared among the various treatment groups to determine whether they are statistically sLJQL¿FDQWO\ GLIIHUHQW 7KH 6WXGHQW¶V W-test was used to compare mean uterine weights between 2 JURXSVDQGDVLJQL¿FDQFHOHYHORIZDVFRQVLGHUHGWREHVWDWLVWLFDOO\VLJQL¿FDQW
Results: Measurement of metabolites in MCF-7 breast cancer cells: Our initial experiments examined whether the enzymes responsible for formation of estrogen metabolites and conjugates were present in human breast cancer cells, as imputed from detection of metabolites (20). MCF-7 cells were incubated with 10 µM 4-OH-estradiol for 24 h before collecting media for later measurement of the various metabolites and conjugates (Figures 3 A-D). As shown in Fig. 3B, we detected 4-methoxy-estradiol as well as the quinone conjugates and the depurinating 4-OHestradiol-1-N7-guanine adduct and its estrone analogue. We next determined whether these cells could aromatize a sufficient amount of testosterone to estradiol to result in formation of the depurinating species. As shown in Fig. 3C, we detected 131 pg/ml of estrogen (92 pg/ml of estradiol and 39 pg/ml of estrone in the media) indicating the production of estrogens from aromatization. The 4-OH-estradiol-1-N7-guanine adduct (and its estrone analogue) was also present at a total concentration of 0.92 pg/ml as were the glutathione, cysteine, and N-acetylcysteine conjugates of estradiol-3,4-quinone and its estrone analogues. Finally, the aromatase inhibitor letrozole was capable of inhibiting the formation of the estrogens from a total of 131 pg/ml of E1 and E2 (Fig. 3C) to 2.8 pg/ml (Fig. 3D) and their downstream metabolites to undetectable levels in most cases. Measurement of metabolites in ERKO/Wnt-1 mammary tissue: Formation of 4-OH-estrogen metabolites is favored over those of the 2-OH species and the catechol-O-methyl-transferase pathway appears to be relatively inactive. We detected 10.9 pmol/g of 4-OH-E2 and 4-OH-E1 in
Author's personal copy mammary tissue, as well as a total of 2.3 pmol/g of conjugated estrogen quinone (Figure 4). No 4methoxy-estrogen metabolites were present. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65
Measurement of metabolites in aromatase transfected mouse mammary gland: As shown in Figure 5, the untreated tissues contained approximately 3pMol/gm amounts of the 3,4 estradiol-adenine adduct. We utilized tetramethoxystilbene (TMS), an inhibitor of CYP 1B1 to inhibit the formation of 4-OH estradiol in these animals. This resulted in marked suppression of the depurinated adduct. Finally, use of the aromatase inhibitor letrozole (CGS) also blocked formation of this adduct.
(5Į-dependent DQG (5 Į-independent effects on tumor development: We postulated that distinction between receptor-dependent and -independent actions might best be elucidated by an experimental animal model using knock-out technology. As shown in Fig. 6, we showed that 50% of mammary tumors arose by 5 months in the ER+ animals versus 11 months in those without ERa (i.e. ERKO animals)(21). This demonstrated WKH PHFKDQLVWLF HIIHFW RI (5Į on the incidence and timing of development of breast tumors in this model. We reasoned that castration, by lowering endogenous E2 and therefore the downstream metabolites, would reduce tumor incidence from levels observed in intact ERKO/Wnt-1 animals. For these experiments, we compared intact and castrate ERKO/Wnt-1 animals. Castration delayed tumor onset (50% incidence time) from 12 to 23 months and reduced tumor incidence from 80% to 50% (p
