© 2000 by Oxford University Press
Journal of the National Cancer Institute Monographs, No. 27, 75-94,
2000
© 2000 Oxford University Press
Chapter 4: Estrogens as Endogenous Genotoxic Agents—DNA Adducts and Mutations
Affiliations of authors: E. Cavalieri, Eppley Institute, University of Nebraska Medical Center, Omaha, NE; K. Frenkel, New York University School of Medicine, NY; J. G. Liehr, Stehlin Foundation for Cancer Research, Houston, TX; E. G. Rogan, Eppley Institute, University of Nebraska Medical Center; D. Roy, University of Alabama at Birmingham, School of Public Health.
Correspondence to: Ercole Cavalieri, D.Sc., Eppley Institute for Research in Cancer and Allied Diseases, 986805 Nebraska Medical Center, Omaha, NE 68198-6805 (e-mail: ecavalie{at}unmc.edu).
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ABSTRACT |
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 TopNotes Abstract IntroductionOncogenic Mutations by...Catechol Estrogen-3,4-Quinones...Estrogen-Mediated Formation of...Is E2 a Genotoxic...ConclusionsReferences |
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Estrogens induce tumors in laboratory animals and have been associated with breast and uterine cancers in humans. In relation to the role of estrogens in the induction of cancer, we examine formation of DNA adducts by reactive electrophilic estrogen metabolites, formation of reactive oxygen species by estrogens and the resulting indirect DNA damage by these oxidants, and, finally, genomic and gene mutations induced by estrogens. Quinone intermediates derived by oxidation of the catechol estrogens 4-hydroxyestradiol or 4-hydroxyestrone may react with purine bases of DNA to form depurinating adducts that generate highly mutagenic apurinic sites. In contrast, quinones of 2-hydroxylated estrogens produce less harmful, stable DNA adducts. The catechol estrogen metabolites may also generate potentially mutagenic oxygen radicals by metabolic redox cycling or other mechanisms. Several types of indirect DNA damage are caused by estrogen-induced oxidants, such as oxidized DNA bases, DNA strand breakage, and adduct formation by reactive aldehydes derived from lipid hydroperoxides. Estradiol and the synthetic estrogen diethylstilbestrol also induce numerical and structural chromosomal aberrations and several types of gene mutations in cells in culture and in vivo. In conclusion, estrogens, including the natural hormones estradiol and estrone, must be considered genotoxic carcinogens on the basis of the evidence outlined in this chapter.
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INTRODUCTION |
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TopNotesAbstractIntroductionOncogenic Mutations by...Catechol Estrogen-3,4-Quinones...Estrogen-Mediated Formation of...Is E2 a Genotoxic...ConclusionsReferences |
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Estrogens, including the natural hormones estradiol (E2) and estrone (E1), induce tumors in various organs of several laboratory animal species and strains [reviewed in (1,2)]. In humans, exogenous estrogen-containing medications or elevated concentrations of circulating endogenous estrogens increase the risk of uterine and mammary cancers [reviewed in (1,2)]. Nevertheless, synthetic or steroidal estrogens or their metabolites failed to induce gene mutations in several classical bacterial and mammalian gene mutation assays (3–7) and were, therefore, classified as epigenetic carcinogens (8,9). Two possible mechanisms of tumor induction by estrogens were subsequently advanced. Estrogen-induced cell transformation and tumor development were proposed to be mediated by 1) estrogen receptor-based proliferation of cells carrying spontaneous replication errors [(8,10); Chapter 8] and 2) disruption of spindle formation and subsequent numeric chromosomal changes (9).
An increasing body of experimental evidence stands, however, in contradiction to these two hypotheses of hormonal tumorigenesis, including the following data: 1) In human mammary epithelial cells, estrogen receptors are expressed in cells different and distinct from proliferating cells carrying proliferation markers (11,12). 2) Aneuploidy and other karyotypic changes were detected in Syrian hamster embryo cells predisposed to immortalization and progression to tumorigenicity; nude mice inoculated with cells carrying such chromosomal alterations, however, did not produce tumors (13). Therefore, additional genetic changes (mutations) were postulated to be required for tumor induction (13). 3) Compared with hamsters treated only with E2, tumor formation is decreased in animals exposed to E2 plus inhibitors of estrogen metabolism (14,15) or to hormonally potent estrogens with poor metabolic conversion to catechol metabolites (16,17). These data support a tumor-initiating role for catechol estrogens (CE). 4) A large body of evidence is accumulating that estrogens induce various types of DNA damage in vitro and in vivo. As outlined below, CE can, indeed, mediate this damage. 5) The classification of estrogens as epigenetic carcinogens is contradicted by preliminary evidence of estrogen-induced gene mutations (reviewed below).
In this chapter, we discuss the induction of DNA damage and gene mutations. First, we focus on the direct adduction of estrogen metabolites to DNA in vitro and in vivo, second on the generation of reactive oxygen species (ROS) by estrogen metabolites and various types of DNA damage induced indirectly by estrogens and, finally, on estrogen-induced gene mutations.
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ONCOGENIC MUTATIONS BY DEPURINATING CARCINOGEN–DNA ADDUCTS AS A MODEL OF ESTROGEN-INDUCED MUTATIONS |
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TopNotesAbstractIntroductionOncogenic Mutations by...Catechol Estrogen-3,4-Quinones...Estrogen-Mediated Formation of...Is E2 a Genotoxic...ConclusionsReferences |
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The origin of cancer represents one of the most intriguing scientific mysteries. Cancer is a disease of mutated critical regulatory genes and abnormal cell proliferation (18). Understanding the origin of these mutations opens the door to strategies for controlling and preventing cancer. One possible approach to investigating the origin of cancer has been to gain a fundamental understanding of the properties of molecules that induce this disease. During the last 25 years, polycyclic aromatic hydrocarbons (PAH) have been investigated by Cavalieri and Rogan (19,20) as model carcinogenic compounds. The purpose of studying these molecules has been threefold: First, they represent a good model for understanding the mechanism of tumor initiation by chemicals; second, they have some geometric resemblance to endogenous estrogens; and third, both PAH and estrogens contain aromatic rings.
Comprehensive studies of PAH have led to an understanding of their mechanism of tumor initiation (19,20). PAH are activated by two main pathways: one-electron oxidation to produce reactive intermediate radical cations and monooxygenation to afford bay-region diol epoxides (19,20). The reactive intermediates formed by these two mechanisms, radical cations and diol epoxides, can bind to DNA to produce adducts that initiate the process of tumor formation, as illustrated in Fig. 1
for dibenzo[a,l]pyrene (DB[a,l]P). DNA adducts are obtained by reaction of the metabolically activated PAH with the nucleophilic groups of the two purine bases, adenine (Ade) and guanine (Gua). These adducts can be either stable or depurinating. The stable adducts are those that remain covalently bonded to DNA unless removed during repair, whereas the depurinating adducts are the ones that are spontaneously released from DNA by destabilization of the glycosidic bond (Fig. 2). Stable DNA adducts are formed when PAH react with the exocyclic amino group of Ade or Gua, whereas depurinating adducts are obtained when PAH covalently bond at the N-3 or N-7 position of Ade or the N-7 or, sometimes, the C-8 position of Gua.
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Among the various approaches to the study of carcinogenesis by PAH, identification and quantitation of their DNA adducts have been the most fruitful in unraveling the mechanism of tumor initiation by these compounds. Through comprehensive studies of the DNA adducts of the potent carcinogenic PAH, benzo[a]pyrene (BP), 7,12-dimethylbenz[a]anthracene (DMBA), and DB[a,l]P (Fig. 3
), Cavalieri and Rogan (20) and Chakravarti et al. (21) have discovered that there is an association between depurinating adducts and oncogenic mutations, suggesting that these adducts are the primary culprits in the tumor initiation process. This discovery was made by identifying and quantifying the DNA adducts formed in mouse skin by BP, DMBA, and DB[a,l]P and, at the same time, determining the mutations in the Harvey (H)-ras oncogene in mouse skin papillomas initiated by these three PAH, as shown in Fig. 4 (21). When mouse skin was treated with DMBA, 79% of the adducts were depurinating Ade adducts and 20% were depurinating Gua adducts (20,22). For DB[a,l]P, 81% were depurinating Ade adducts and 18% were depurinating Gua adducts (20). In contrast, mouse skin treated with BP produced 46% depurinating Gua adducts and 25% depurinating Ade adducts (20,23). Examination of the ras oncogene mutations in papillomas induced by DMBA or DB[a,l]P demonstrates that, in both cases, an A 
T transversion (CAA CTA) consistently occurs (Table 1; Fig. 5) (21). These mutations associate with the predominant formation of depurinating Ade adducts by these two PAH. About twice as many of the papillomas induced with BP contain G T mutations at codon 13 in ras (GGC GTC) compared with the number of tumors with a codon 61 CAA CTA mutation (21,24). The ratio of mutations is consistent with the profile of depurinating Gua and Ade adducts formed by BP in the target tissue (Table 1). This pattern of ras mutations suggests that the oncogenic mutations in mouse skin papillomas induced by these PAH are generated by misreplication or misrepair of the apurinic sites derived from loss of the depurinating adducts (21). For example, an A T transversion can be attributed to loss of a depurinating Ade adduct and generation of an apurinic site. If the apurinic site is not correctly repaired in the next round of DNA replication, the most likely base to be inserted opposite the apurinic site is Ade (Fig. 6). When the coding strand of the DNA is then replicated, a thymine is inserted opposite the new Ade, resulting in the A T mutation observed in codon 61 of the ras oncogene in tumors initiated by PAH forming predominantly depurinating Ade adducts. When a Gua adduct is lost by depurination, leaving an apurinic site in the DNA, the preferential insertion of Ade in the opposite DNA strand leads to a G T transversion at the site of the adduct.
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It is also possible that the ras mutations are generated by misrepair, rather than misreplication, of the apurinic sites. Strong evidence for misrepair is provided by the observation of codon 61 CAA
CTA transversions in mouse skin DNA 1 day after treatment with DB[a,l]P (Table 2) (25), when the cells are unlikely to have divided. The A T transversions are present in 0.1% of the cells by day 1, increase to about 5% by day 3, and then decrease to background levels by day 9. Subsequently, the A T mutation is detected in increasing levels as papillomas begin to develop.
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Because thousands of apurinic sites are spontaneously formed per cell each day, repair of apurinic sites induced by PAH might be expected. The level of apurinic sites arising from treatment with PAH is, however, 15–120 times higher than those formed spontaneously, suggesting that this large increase in apurinic sites could overwhelm the capacity of the cell to repair them before replication occurs (20,21). Furthermore, the apparent nonrepair of apurinic sites induced by treatment with PAH may also be due to the presence of stable adducts that could interfere with error-free repair of apurinic sites. Thus, apurinic sites can generate the mutations that play the critical role in the initiation of cancer, and formation of depurinating adducts has become the common denominator for recognizing the potential of a chemical to initiate cancer.
The evidence that depurinating PAH–DNA adducts play a major role in tumor initiation has provided the impetus for discovering the estrogen metabolites that form depurinating DNA adducts and can be potential endogenous initiators of many human cancers (26,27).
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CATECHOL ESTROGEN-3,4-QUINONES AND APURINIC SITES IN CANCER INITIATION |
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TopNotesAbstractIntroductionOncogenic Mutations by...Catechol Estrogen-3,4-Quinones...Estrogen-Mediated Formation of...Is E2 a Genotoxic...ConclusionsReferences |
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CEs are among the major metabolites of E1 and E2, as discussed in Chapter 5. If these metabolites are oxidized to catechol estrogen quinones (CE-Q), they may react with DNA to form depurinating adducts. It is hypothesized that these adducts generate apurinic sites leading to mutations, which may initiate breast, prostate, and possibly other human cancers. The estrogens E1 and E2 are biochemically interconvertible by the enzyme 17
-estradiol dehydrogenase. These two estrogens are metabolized via two major pathways: formation of CE (Fig. 7) and, to a lesser extent, 16
-hydroxylation (not shown). The catechols formed are the 2-hydroxylated and 4-hydroxylated estrogens (28,29). Generally, these two CEs can be inactivated by O-methylation catalyzed by catechol-O-methyltransferases (COMT) (28). Other possible conjugations of CE, such as glucuronidation and sulfation (not shown), may also play a role in inactivation/protection (Chapter 6). If formation of the 4-hydroxylated metabolites is excessive (see below) and/or production of these methyl, glucuronide, or sulfate conjugates is insufficient and, thus, the cells are not totally protected from CE toxicity, competitive catalytic oxidation to semiquinones (CE-SQ) and CE-Q can occur. CE-SQ and CE-Q may conjugate with glutathione (GSH), catalyzed by S-transferases. If this inactivating process is incomplete, CE-Q may react with DNA to form stable and depurinating adducts (26,27).
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To determine the possible genotoxic effects of CE-Q, they were reacted with the nucleosides 2'-deoxyguanosine (dG) and 2'-deoxyadenosine (dA) and the nucleobase Ade (26,30). An acetonitrile solution of E1 (E2)-3,4-Q was mixed with dG, dissolved in acetic acid/water (1 : 1) (Fig. 8
). The adduct 4-OHE1(E2)-1(,)-N7Gua was formed during 5 hours at room temperature (26), and it is a mixture of two conformational isomers resulting from the restricted rotation of the Gua moiety around the N7(Gua)-C1(estrogen) bond. The reaction of the CE-3,4-Q with dG at the N-7 position destabilizes the glycosidic bond and results in loss of the deoxyribose moiety. When the adduct is formed by reaction of CE-3,4-Q with DNA, it is released from the DNA by spontaneous depurination. Reaction of E1(E2)-3,4-Q with dA produced no adducts; however, reaction of E1(E2)-3,4-Q with Ade resulted in the formation of 4-OHE1(E2)-1(,)-N3Ade (Fig. 8) (30). This adduct was obtained only with Ade because in dA the adjacent deoxyribose bonded to N-9 impedes the approach of the electrophile E1(E2)-3,4-Q to N-3 (23,31). This interference is not present in DNA, as evidenced by formation of PAH-N3Ade adducts, which are rapidly lost from the DNA by depurination (23,32).
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When E1-2,3-Q reacted with dG or dA, a profile of adducts totally different from those formed by E1-3,4-Q was obtained (Fig. 9
) (26). Reaction of E1-2,3-Q with dG afforded 2-OHE1-6-N2dG and with dA yielded 2-OHE1-6-N6dA. In this case, the E1-2,3-Q did not react as a quinone, but as its tautomer, the E1-2,3-Q methide. This electrophile reacts at C-6 with the exocyclic amino group of dA or dG to yield the N6dA and N2dG adducts, which retain the deoxyribose and are referred to as "stable" adducts because they remain in DNA unless repaired.
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To determine whether these adducts are formed in biologic systems, E2-3,4-Q or enzymically activated 4-OHE2 was reacted with DNA for 2 hours at 37 °C (Fig. 10
). The stable adducts were determined by the 32P-postlabeling method, and depurinating adducts were analyzed by high-performance liquid chromatography (HPLC) interfaced with an electrochemical detector (27). When E2-3,4-Q reacted with DNA, almost the same amount of 4-OHE2-1(,)-N7Gua and 4-OHE2-1(,)-N3Ade were obtained (Table 3). The amount of stable adducts was 0.02% of the depurinating adducts. Activation of 4-OHE2 with horseradish peroxidase gave similar results, whereas lactoperoxidase produced a similar amount of N3Ade adduct but about 50% more N7Gua adduct (Table 3). The same two depurinating adducts were obtained in equal but smaller amounts when 4-OHE2 was activated with phenobarbital-induced rat liver microsomes (cytochrome P450) (27).
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When female Sprague–Dawley rats were treated by intramammillary injection of 4-OHE2 or E2-3,4-Q, the 4-OHE2-1(
,)-N7Gua adduct was detected in the mammary tissue (27). The N3Ade adduct presumably was also present, but its synthetic standard was not available at the time of the study. These data clearly show that CEs are enzymatically oxidized to CE-Q and bind to DNA in vitro and in vivo. Several additional lines of evidence suggest that oxidation of 4-hydroxyestrogens is the pathway leading to estrogen-induced cancer. 4-Hydroxyestrogen formation has been observed to predominate in hamster kidney (33,34) and other organs prone to estrogen-induced tumors, such as rat pituitary (35) and mouse uterus (36). In fact, 4-hydroxyestrogens induce kidney tumors in male Syrian golden hamsters, whereas the 2-hydroxyestrogens do not (4,37). Predominant 4-hydroxylase activity has also been found in human microsomes of uterine myometrium and benign uterine leiomyomas (38) as well as in microsomes of benign and malignant breast tumors (39,40). In tissues resistant to estrogen-induced tumors, such as the liver, formation of 2-hydroxyestrogens predominates (39). Furthermore, 2,3,7,8-tetrachlorodibenzop-dioxin induces cytochrome P450 1B1, which predominantly catalyzes hydroxylation of E2 at the C-4 position (41–44). The importance of this finding is related to evidence that exposure to dioxin greatly increases the risk of developing cancer (42,45). The combination of increased formation of the 4-hydroxylated CEs and their oxidation to CE-3,4-Q, which react with DNA to form the depurinating adducts associated with oncogenic mutations, suggests that the 4-hydroxyestrogen pathway producing CE-3,4-Q is responsible for the genotoxic effects leading to estrogen-induced initiation of cancer.
The kidney of male Syrian golden hamsters is an established model for estrogen-induced tumorigenesis (46,47). Two hours after hamsters were given an injection intraperitoneally with 4-OHE2, the kidneys were removed and extracts were analyzed for the adducts formed by 4-OHE2 with DNA and GSH by using HPLC with electrochemical detection and confirmation by mass spectrometry (48). With DNA, the predominant adducts are 4-OHE2-1(,)-N7Gua and 4-OHE2-1(,)-N3Ade; only the N7Gua adduct was analyzed because different gradient conditions would have been needed for the N3Ade adduct. With GSH, 4-OHE2 forms the 4-OHE2-2-SG conjugate (49,50). This conjugate is further metabolized to 4-OHE2-2-cysteine and 4-OHE2-2-N-acetylcysteine by the mercapturic acid biosynthesis pathway. Therefore, all of these conjugates were searched for, along with the 4-OHE2-1(,)-N7Gua adduct. Preliminary results indicate that 4-OHE2-1(,)-N7Gua and all of the GSH-derived conjugates are present in the kidney 2 hours after injection of 4-OHE2, with the cysteine conjugates being the most abundant. As hypothesized for the initiation of cancer by estrogens, these results demonstrate that, in the hamster kidney, 4-OHE2 is oxidized to E2-3,4-Q, which binds to GSH and to DNA, forming depurinating adducts.
The nonsteroidal estrogen hexestrol, which is diethylstilbestrol (DES) hydrogenated at the C-C double bond, is carcinogenic in Syrian golden hamsters (46,51). The major metabolite of hexestrol and DES is their catechol (51–54), which can be metabolically converted to their catechol quinone. This hexestrol quinone has chemical and biochemical properties similar to those of CE-3,4-Q, i.e., it specifically forms an N7Gua adduct after reaction with dG or DNA (55). These data suggest that the hexestrol catechol quinone is the electrophile involved in tumor initiation by hexestrol. In turn, these results substantiate the hypothesis that CE-3,4-Q may be endogenous tumor initiators.
In conclusion, the pathway of activation, i.e., oxidation of estrogens to CE and then to CE-Q, affords the ultimate carcinogenic metabolites that are CE-3,4-Q for endogenous estrogens and catechol quinones for synthetic estrogens. This competitive, oxidative pathway takes place only when excessive formation of 4-CE and/or their incomplete inactivation occur. The DNA damage by these reactive electrophiles consists of the formation of depurinating adducts and apurinic sites in DNA. Misrepair and/or misreplication of the apurinic sites in DNA may generate the critical mutations that trigger induction of cancer by estrogens (21,25).
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ESTROGEN-MEDIATED FORMATION OF OXIDANTS AND OXIDATIVE DNA DAMAGE: THEIR ROLE IN CARCINOGENESIS |
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TopNotesAbstractIntroductionOncogenic Mutations by...Catechol Estrogen-3,4-Quinones...Estrogen-Mediated Formation of...Is E2 a Genotoxic...ConclusionsReferences |
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Oxidants are continuously formed and degraded in normal cellular processes. They are necessary for a plethora of biochemical reactions, without which life itself could not be sustained. Cells are equipped with extensive multilayer antioxidant defenses to intercept excess oxidants. However, when ROS are generated at an inappropriate time, in excessive amounts, or when antioxidant defenses are overwhelmed, then the negative effects of oxidants become apparent. In the following presentation, we will concentrate on the damaging effects of oxidants induced endogenously by estrogens and their putative role in the carcinogenic process.
One of the major types of damage that oxidants directly induce is oxidative modification of the genetic material. There are well over 30 different types of oxidized bases that can be formed in DNA; their levels exceed those of the stable carcinogen-induced adducts with DNA bases by about two orders of magnitude, being on average 1/105 versus 1/107 normal DNA bases, respectively (56,57). Some of these oxidized DNA bases are mutagenic (58–60) and induce DNA hypomethylation (61,62), a process known to increase gene expression (63).
The oxidized base derivatives most frequently discussed in this presentation are 8-hydroxy-2'-deoxyguanosine (8-OHdG) and 5-hydroxymethyl-2'-deoxyuridine (HMdU) (for structures see Fig. 11). As oxidized bases are formed in DNA, various types of repair enzymes start removing them (64,65). There is always a background level of oxidized bases present in DNA, which seems to be tolerated by the cells. However, conditions leading to a continuous elevation of oxidized bases in DNA are the same as those that induce tumor promotion processes.
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The following examples illustrate that target sites for estrogen carcinogenesis invariably also contain elevated levels of oxidized bases in cellular DNA. Conversely, the presence of higher than normal amounts of oxidized DNA bases may be indicative of a carcinogenic process induced by estrogens.
Oxidized DNA Bases as Evidence of Endogenous Oxidant Formation by Estrogens
Animal models.
The formation of either 8-OHdG or HMdU in the target tissues for estrogen-mediated carcinogenesis has repeatedly been shown. The animal models often utilized are Syrian hamster kidney (66) and dorsolateral prostate in the Noble rat [(67); M. Bosland: unpublished data]. For example, the highest 8-OHdG increase (sevenfold) occurred in the periurethral section of the dorsolateral prostate isolated from the Noble rat treated with testosterone and E2 (Table 4; M. Bosland: unpublished data). This is the same part of the organ where adenocarcinoma growth (83% of animals), DNA adduct formation (
fourfold increase), and lipid peroxidation (>threefold) occur (Chapter 2). There was no cancer growth, DNA adducts, or 8-OHdG formation in the periphery. Thus, the oxidative DNA damage was detected at the same selected tissue site (10% of the whole prostate) where adenocarcinoma develops.
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Formation of 8-OHdG and HMdU was also significantly elevated and persisted in mouse skin topically treated with DMBA, a potent skin and mammary carcinogen, through the stages of tumor promotion and progression, and was evident at the time of tumor growth (Fig. 12
) (68). The importance of oxidants and oxidative DNA damage in DMBA carcinogenesis is underscored by the long-known fact that pretreatment with antioxidants causes a suppression of DMBA-induced tumors without affecting levels of stable DNA adducts (69). DMBA treatment evoked sustained, long-lasting inflammatory responses characterized by neutrophilic infiltration and edema (68). DMBA also enhances expression of IL-1 messenger RNA (mRNA) and elevates the activity of IL-1 (70). Estrogens, even at a low physiologic dose, also increase formation of this inflammatory cytokine (71), which, in turn, has a pronounced effect on a cascade of further inflammatory and carcinogenic responses (72–74).
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Cellular models. Both HMdU and 8-OHdG are present in MCF-10A human breast epithelial cells (immortal but not tumorigenic) and in MCF-7 breast cancer cells (75,76). The basal levels of both modified bases are
80% higher in MCF-7 cells, a finding that is expected because tumor cells produce substantial levels of hydrogen peroxide (H2O2), one of the major cellular oxidants (57,77,78). HMdU levels increased in response to H2O2 (75), and those of 8-OHdG, in response to the DMBA treatment (76). These increases are higher in MCF-10A cells and reach levels prevalent in tumor cells. Carcinogen treatment of MCF-10F cells (also immortal but otherwise a normal cell line) is known to lead to their malignant transformation (79). Hence, the increase in oxidized DNA bases likely occurs during the process of cellular transformation. Humans. More important, HMdU was shown to be present in white blood cell DNA of women at a high risk for breast cancer and those diagnosed with breast cancer (80,81). Of interest, a decrease in fat intake and presumed increase in vegetables and fruit consumption significantly decreased HMdU levels in women at high risk for breast cancer. In general, levels of oxidized purines were significantly elevated in human breast cancer. However, they were also increased even at sites distal to the cancerous tissue, not only in the breast, but also at other sites of hormonal carcinogenesis, such as ovarian and, in men, prostatic cancers [(82,83); Chapter 9]. In fact, it has been proposed that the increased levels of oxidized bases in human DNA precede cancer development and may serve as biomarkers of cancer risk (81,83,84). There is extensive evidence that chemical carcinogens generally induce formation of oxidized bases in DNA of target tissues in vivo. Estrogens are no exception and induce tumors by comparable mechanisms (57,66,85).
Mechanisms of Estrogen-Mediated Oxidant Formation
What are the sources of endogenous oxidants formed in response to estrogens? Estrogens, like other chemical carcinogens, are metabolized by cytochrome P450 enzymes and form hydroxylated products. The main metabolites of E2 include 2-, 4-, and 16-hydroxyestradiol (Fig. 13) (33–36,44,86–88). The 2- and 4-hydroxylated catechols contain the hydroxyl groups in a vicinal position, which predisposes them to further oxidation. Both can be oxidized to semiquinones, which in the presence of molecular oxygen are oxidized to quinones with formation of superoxide anion radicals (O •2- ), as illustrated in Fig. 7
(66,89,90). These O •2- readily dismutate to H2O2 either spontaneously or even faster when catalyzed by superoxide dismutase. H2O2 is neutral and rather nonreactive, except in the presence of the reduced transition metal ions (i.e., Fe2+ and Cu+), which cause formation of the most powerful and indiscriminate oxidants, hydroxyl radicals (•OH) (91,92). However, as a neutral molecule, H2O2 can readily cross the cellular and nuclear membranes and reach DNA in neighboring cells, where it can cause site-specific oxidation of bases (57). Quinones and semiquinones are capable of redox cycling as long as there is molecular oxygen available and, therefore, even a small amount of E2 may cause substantial ROS production and subsequent cellular damage.
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This ROS formation by redox cycling of semiquinones and quinones is mitigated by cellular quinone reductase, an enzyme that reduces quinones back to catechols by use of using reduced nicotinamide adenine dinucleotide (NADH) as a reducing cofactor. Moreover, COMT may prevent oxidation of CE to CE-SQ by methylating 2- or 4-hydroxyl groups (Fig. 14
). However, it appears that the 4-hydroxyl group is not as readily methylated as is the 2-hydroxyl substituent, which results in the predominance of 4-OHE2 in redox cycling, while 2-OHE2 is virtually inactivated by methylation (93). Furthermore, methylated 2-OHE2 was shown to inhibit COMT-mediated methylation of 4-OHE2, which may allow for the accumulation of this carcinogenic metabolite in those organs where both metabolites are formed (93). The rapid methylation of 2-OHE2 may be one reason for its lack of carcinogenic activity (4,37), whereas the lesser methylation of 4-OHE2 contributes to its carcinogenic properties (4,37,39).
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Like PAH, estrogens can generate ROS by peroxidatic metabolism (94). For example, E2 was shown to produce phenoxyl radicals in the lactoperoxidase-catalyzed reaction. These phenoxyl radicals rapidly react with the cellular-reducing agents GSH and NADH. However, instead of detoxification, other radical species are formed (GS• and NAD•, respectively), which reduce molecular oxygen to O •2- , followed by H2O2 formation (Fig. 15
). The regenerated GSH and NADH can continue this process as long as O2 is available. Of the cellular reductants tested, only ascorbate radical does not further react with O2, thereby breaking the radical chain that leads to ROS formation. Lactoperoxidase and estrogens are ubiquitously present in milk ducts and in the mammary gland (94).
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Estradiol-Induced Lipid Peroxidation and DNA Damage
ROS may also cause oxidation of cellular macromolecules other than DNA, which include proteins and lipids. For example, oxidation of cysteine residues at an active site of an enzyme would either inactivate or at least change the activity of that enzyme (95). Many biosynthetic and energy-producing antioxidants as well as repair enzymes have redox-sensitive centers, which are readily modified by prooxidant changes (96). Hence, E2-induced ROS may have a pronounced effect on cell maintenance and functioning.
Lipids, particularly polyunsaturated lipids, are readily peroxidized, and the products often participate in a chain reaction propagating formation of various radical species (97). Lipid hydroperoxides are formed during prooxidant conditions generated by different sources, which include inflammation and carcinogen exposures. Again, like other carcinogens, estrogens induce lipid peroxidation during their metabolic activation (66). The insidiousness of this process is demonstrated by the fact that lipid hydroperoxides formed during E2 metabolism may serve as cofactors in further E2 (or other carcinogen) metabolism to hydroxylated products and in the oxidation of CE to quinone intermediates, which continuously amplifies the formation of lipid hydroperoxides and cellular damage (Fig. 16). Furthermore, lipid hydroperoxide-derived aldehydes, such as malondialdehyde and 4-hydroxynonenal, interact with bases in cellular DNA, thus increasing the burden of DNA modification (98,99).
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An interplay between estrogen metabolites and oxidants leads to at least three types of DNA base damage (Fig. 16
): DNA base adducts produced by quinones, as described above, lipid hydroperoxide-derived aldehyde DNA adducts, as well as a plethora of oxidized DNA bases. The importance of ROS formation during estrogen metabolism is underscored by the fact that H2O2 generated by the redox cycling of the semiquinone-quinone couple is readily reduced by cellular transition metal ions, such as Fe2+ and Cu+, to hydroxyl radicals (•OH), the most potent oxidants. Hydroxyl radicals not only oxidize bases in DNA, but also cause lipid peroxidation. Lipid hydroperoxides then serve as cofactors in further estrogen metabolism, which leads to additional semiquinone–quinone redox cycling, ROS production, and so on. Estradiol-Mediated Modulation of Immune Responses
Although estrogens themselves can induce ROS production, they can also modulate immune responses and immune-mediated diseases, as indicated in Table 5, which can predispose to cancer (71,100). This process may take place under prooxidant conditions occurring in the course of inflammatory processes. In fact, at physiologic doses, E2 potently induces interleukin (IL)-1, a cytokine that can initiate a cascade of other cytokines, chemotactic and growth factors (71,101). Chemotactic factors cause infiltration of phagocytes, which may be activated to secrete a plethora of other cytokines, ROS, and reactive nitrogen species (RNS) (72,74,102–105). On the other hand, E2 inhibits IL-1-induced IL-6 production. Therefore, by suppressing IL-6 formation, E2 increases human epithelial cell proliferation, a process important in tumor growth, while it also inhibits the activity of natural killer cells, thus allowing tumor growth (101,105). E2 mediates macrophage proliferation and decreases cell differentiation (71). Each of the affected processes contributes to the environment, allowing or encouraging tumor cell development and growth.
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Estrogen Effects on Macrophages and Their Production of ROS and RNS
Macrophages have been found in normal human breast tissue. However, their numbers increase tremendously in breast tumors, providing up to 50% of the tumor mass (106,107). This increase in mass might be compounded by estrogen-stimulating macrophage proliferation (71). Substantial levels of estrogen are produced from androgens within the female breast by the action of aromatase, a cytochrome P450 enzyme that is present in cells as well as in macrophages (88,108,109). Thus, estrogens cause macrophage proliferation and activation and, in turn, macrophages produce estrogens, which may act on other phagocytic cells, and so on.
On stimulation, macrophages produce oxidants such as O•2- and H2O2 by an reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase catalyzed reduction of molecular oxygen. This process occurs rapidly by the activation of the constitutive NADPH oxidase. A few hours after macrophage stimulation, inducible nitric oxide synthase is synthesized by the cells and mediates production of L-arginine-derived nitric oxide (•NO), another radical species, which participates in signal transduction, numerous reactions, and cellular processes. O •2- and •NO may rapidly interact, with the evolution of peroxynitrite, a much more potent oxidant (110). Hence, macrophage activation may lead to ROS and RNS, which include O •2- , H2O2, •OH, and singlet oxygen [1O2, known to oxidize dG to 8-OHdG (111)], peroxynitrite (ONOO-), nitrite, nitrate, as well as nitrating species. Among them, ROS and RNS may cause DNA base hydroxylation, oxidation, nitration, and deamination (111–113).
Estrogen Effects on PMN Function and ROS Formation
Estrogens also affect the function of PMNs (polymorphonuclear leukocytes, neutrophils, granulocytes), which are another group of phagocytic cells that produce copious amounts of ROS on their stimulation. In addition to NADPH oxidase, PMNs express myeloperoxidase, an enzyme that catalyzes oxidation of chloride ions by H2O2 (generated by NADPH oxidase) to hypochlorite/hypochlorous acid (HOCl/OCl-), one of the most potent oxidants (57). This reaction is catalyzed by myeloperoxidase released from PMNs during their activation process. HOCl/OCl- is utilized as a bacteriocidal and tumoricidal agent within the organism. However, estrogens and some of their metabolites (i.e., E2, E1, 16-OHE1, and estriol) may induce myeloperoxidase release from the resting (inactivated) cells and stimulate generation of oxidants in the absence of pathogens (114). Of interest, 2-hydroxylated estrogens act as powerful inhibitors of PMNs activity, possibly one of the protective properties of the 2-hydroxylated CE.
The estrogen-mediated action causes HOCl/OCl- formation and ensuing oxidative cell damage, even in the absence of the proper targets. Moreover, estrogens can stimulate (by 10-fold) the activity of the released myeloperoxidase, thus compounding the damaging effects. The interaction of HOCl/OCl- with an excess of H2O2 causes regeneration of chloride ions as well as evolution of species that can chlorinate and oxidize DNA (Fig. 17) (112,114).
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Various interactions occur at physiological pH among reactive oxygen and nitrogen species generated by the phagocytic cells, macrophages and PMNs (Fig. 17
). Those interactions lead to various types of DNA modification, many of which result in mutations. Therefore, estrogens, by modulating phagocytic cell proliferation and activation, have a pronounced effect on the integrity of DNA and mutagenesis.
ROS generated by PMNs and macrophages cause not only DNA modification but also oxidation of proteins and lipid peroxidation. As shown in Fig. 16, lipid hydroperoxides can now serve as cofactors of estrogen metabolism, during which ROS are produced, as well as other DNA-damaging species. Therefore, estrogens affect inflammatory responses and, in turn, their activities are affected by the inflammation products.
Immunomodulation by Estrogens
One of the more pronounced properties of E2 is its ability to differentiate T and B cells, increase immunoglobulin production, and aggravate immune complex-mediated diseases, such as systemic lupus erythematosus, which occur predominantly in females (115–117). This disease is characterized by strong inflammatory responses, which are thought to contribute to it, as well as to various types of cancer (57,73,112,117).
Women at high risk of breast cancer, those with benign breast diseases, and those who are diagnosed with breast cancer years later have significantly elevated anti-HMdU autoantibodies (84). As Fig. 18 shows, the levels of anti-HMdU autoantibodies are remarkably stable over a period of years. The presence of high anti-HMdU autoantibody levels attests to the prooxidant conditions that have led to oxidation of bases in cellular DNA and have evoked an autoimmune response. These results also suggest that the oxidative DNA base damage (HMdU) and the biologic responses it evokes (anti-HMdU autoantibodies) start occurring early in the carcinogenic process. Such a conclusion is strengthened by data obtained by other investigators, who showed that oxidative DNA base damage is evident in DNA of individuals at risk for hormone-dependent cancers (81,84,118).
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Effect of Estrogens on Oncogenes and Transcription Factors
E2 was shown to induce macrophages to produce immediate early genes c-fos and c-jun, and the AP-1 transcription factor, which is a heterodimer of these two genes (71). AP-1-binding sites are present in promoter regions of many growth factors as well as antioxidant enzymes (112,119,120). E2 also induces c-myc, an oncogene known to be important in tumor promotional processes, and bcl-2, a gene inhibiting apoptosis. Oxidants are known to induce all of these oncogenes, whereas antioxidants counteract their formation (57). Thus, E2 through its ROS-inducing capabilities affects processes leading to mutations, governing tumor growth, and tumor surveillance.
Effects of estrogens on oxidant formation, oxidative DNA damage, and other cellular processes, which contribute to the carcinogenic properties of estrogens, are summarized in Table 6. Most of these effects are exactly the same as those induced by other chemical carcinogens.
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Tamoxifen as an Anticarcinogen
Tamoxifen has been used as a drug that effectively prevents recurrence of breast cancer. It has also been recently shown to decrease substantially breast cancer in women at high risk for this disease based on a strong family history. The controversy over its use as a preventive agent exists because tamoxifen increases the rate of endometrial cancer in susceptible individuals (121).
Tamoxifen exerts its antiproliferative effects on the estrogen receptor-positive cells as well as on cells lacking that receptor (122,123). This suggests that the mode of action of tamoxifen in suppressing breast cancer relies not only on its antiestrogenic properties but must involve effects on other factors important in the carcinogenic process. Tamoxifen has been shown to be a potent antitumor promoter in animal models, while estrogens can act as tumor promoters. Many of the processes known to contribute to tumor promotion are inhibited by tamoxifen (Table 7) (124). These include inhibition of hyperplasia, inflammation, ROS production, oxidative DNA base damage, and lipid peroxidation. Estrogens induce all of these processes. Hence, the fact that tamoxifen, an antibreast cancer drug, suppresses so many of the estrogen-induced prooxidant processes and factors strengthens the hypothesis of estrogen being a carcinogen that acts through elevation of oxidative stress. The interrelationships among many factors contributing to estrogen-induced oxidative stress and carcinogenesis are summarized in Fig. 19.
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IS E2 A GENOTOXIC OR EPIGENETIC CARCINOGEN? |
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TopNotesAbstractIntroductionOncogenic Mutations by...Catechol Estrogen-3,4-Quinones...Estrogen-Mediated Formation of...Is E2 a Genotoxic...ConclusionsReferences |
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Pharmacologic levels of estrogens are known to produce toxic effects, such as embryotoxicity, teratogenicity, and carcinogenicity (2,125,126). The molecular mechanism(s) by which estrogens cause such adverse effects are under investigation from several different angles. Recent epidemiologic and laboratory findings have increased the growing concern that instability in the genome induced by estrogens may be involved in the induction of certain types of cancer in humans (127). Estrogen-induced genotoxicity is an important contributor to the induction of toxic effects, because estrogen receptor-mediated events by themselves cannot explain the carcinogenic and noncarcinogenic adverse properties of estrogens. The lack of mutagenic activity in bacterial and mammalian cell mutation assays (3–7) led to estrogens being categorized as nongenotoxic and nonmutagenic chemicals (8,9,128). However, more recent results, such as the arrest of DNA replication deriving from estrogen–DNA adducts, the enhancement of homologous recombination, and mutations in individual genes and in microsatellite repeat sequences clearly indicate that estrogens are able to induce multiple types of genetic insults in cells. This part of the chapter will focus on estrogen-mediated damage at the genome level leading to the development of mutations.
Indirect Evidence of Mutations Induced by Estrogens
Mutations may result from numeric changes or structural alterations in the genome. These include extra or missing copies of microsatellite DNA, transcriptional silencing, chromosomal deletions, frameshifts, amplifications, rearrangements, translocations, and other changes that interfere with the integrity of the genome. More recent experiments have shown that some estrogens are capable of producing such instability (126,129,130). For instance, estrogens induce numeric changes in chromosomes (genome mutation or aneuploidy) with and without apparent DNA damage (131). Both DES and E2 are potent inhibitors of mitosis in vitro and are capable of inducing genomic mutations in cultured cells (132). Potential targets for numeric changes in chromosomes are the spindle apparatus (microtubules and centrioles), DNA, regulating proteins, and centromere. Chromosomal analysis of tissues of mice exposed perinatally to estrogen demonstrates that this treatment induces chromosomal aberrations in the same target tissues in which tumors subsequently develop (133,134). DES or E2 treatment of hamsters produces renal chromosomal aberrations, including deletions, inversions, and translocations (135,136).
DNA damage either by free radicals or by genotoxic reactive metabolites is known to cause structural changes in chromosomes. Formation of oxygen-free radicals by redox cycling of estrogens or DNA modification by reactive estrogen metabolites may explain some of the structural and numeric chromosomal changes observed in response to estrogen exposure (131,132). Damage to DNA by ROS generated by estrogen treatment, i.e., 8-OHdG, lipid-DNA adducts, and DNA strand breaks, may induce structural and numeric alterations in chromosomes and may be important lesions capable of producing mutagenic changes in the genome.
Direct Evidence of Gene Mutations Induced by Estrogens
The potential of estrogens to induce mutations has been highly controversial. Previous studies of the mutagenic potential of estrogens showed that neither they nor their reactive intermediates induced mutations in the Ames bacterial reversion test or in Syrian hamster embryo cells (3–7). However, these results are not consistent with the covalent binding of reactive estrogen metabolites to bases of DNA or with the ROS-mediated changes to DNA bases, as discussed above. Both of these types of DNA lesions are capable of inducing mutations. Some earlier studies showed that DES and E2 can increase mutations leading to ouabain resistance (137). Experiments demonstrate that DES quinone increases homologous recombination in Escherichia coli (138). Both DES and E2 are mutagenic in the gpt+ Chinese hamster G12 cell line (7).
Re-examination of the mutagenic potential of E2 at various concentrations demonstrated a weak, but detectable, mutagenicity of E2 at the lowest dose assayed (10-10 µM E2) in V79 Chinese hamster lung cells [(7); Albrecht T, Liehr JG: unpublished data]. Covalent DNA adducts formed by DES quinone and CE quinone arrest the progression of cytochrome oxidase III gene synthesis (139). The mRNA for the repair enzyme DNA polymerase obtained from DES-induced kidney tumors carries several mutations in the catalytic domain compared to that of age-matched control kidney (140). Kidney tumors and premalignant kidney of E2-treated hamsters contain mutations in repeat sequences of microsatellite DNA (141,142). Recently, mutational changes in an unidentified gene have been observed in the stilbene-induced hamster kidney tumor (Singh LP, Roy D: unpublished data). A high frequency of genomic rearrangements have been observed in transformed 10T1/2 mouse cell subclones treated with E2, indicating that this and other natural hormones may accelerate the accumulation of mutations (143).
Genetic instability manifested by somatic mutation of microsatellite repeats occurs with high frequency in clear cell adenocarcinomas of the vagina and cervix, with evidence of microsatellite instability in all DES-associated tumors examined (144). Furthermore, mutations have been reported in the p53 gene at codons 274 and 140 in hepatocellular carcinoma associated with use of oral contraceptives (145). A good association has been shown between induction of aneuploidy, DNA adducts, and estrogen-induced cell transformation (146). These findings indicate that several types of mutations induced by estrogens may not be detectable by the Salmonella reversion test or assay of gene mutations at specific narrowly defined loci in Syrian hamster embryo cells. Taken together, these findings suggest that estrogens can produce multiple types of genetic insults contributing to the induction of genomic instability. Several structural and numeric changes have already been demonstrated at the cellular level in response to DES or E2 exposure (147).
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CONCLUSIONS |
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TopNotesAbstractIntroductionOncogenic Mutations by...Catechol Estrogen-3,4-Quinones...Estrogen-Mediated Formation of...Is E2 a Genotoxic...ConclusionsReferences |
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In this chapter, we have outlined a large body of evidence that estrogens, including the natural hormones E2 and E1, damage genetic material in various ways. Direct estrogen DNA adducts and/or indirect forms of covalent DNA alterations may be induced in experimental systems in vitro, cells in culture, and laboratory animals or may be detected in humans. Study of model carcinogenic PAH has led to the discovery that apurinic sites in DNA generated by depurinating adducts can lead to the mutations that trigger the cancer process. The CE metabolites, when oxidized to the electrophilic CE-Q, may react with DNA to form stable and depurinating adducts. The 4-CE that form predominantly depurinating adducts are carcinogenic, whereas the noncarcinogenic 2-CE exclusively form stable DNA adducts. Oxidation of CE also leads to overwhelming amounts of ROS that generate extensive DNA damage and contribute to tumor promotion.
Preliminary data also suggest that estrogens, including the natural hormones E2 and E1, may induce gene mutations. The more recent gene mutation experiments reported in this chapter are compatible with the lack of mutagenic activity