TY - JOUR
AU - Roy, Deodutta
AB - Abstract 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. 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. Oncogenic Mutations by Depurinating Carcinogen–DNA Adducts as a Model of Estrogen-Induced Mutations 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. 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. 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. 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). Catechol Estrogen-3,4-Quinones and Apurinic Sites in Cancer Initiation 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 17β-estradiol dehydrogenase. These two estrogens are metabolized via two major pathways: formation of CE (Fig. 7) and, to a lesser extent, 16α-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). 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(α,β)-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(α,β)-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). 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. 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(α,β)-N7Gua and 4-OHE2-1(α,β)-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). When female Sprague–Dawley rats were treated by intramammillary injection of 4-OHE2 or E2-3,4-Q, the 4-OHE2-1(α,β)-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(α,β)-N7Gua and 4-OHE2-1(α,β)-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(α,β)-N7Gua adduct. Preliminary results indicate that 4-OHE2-1(α,β)-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). Estrogen-Mediated Formation of Oxidants and Oxidative DNA Damage: Their Role in Carcinogenesis 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. 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. 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-1α messenger RNA (mRNA) and elevates the activity of IL-1α (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). 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 16α-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. 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). 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). 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). 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)-1α, 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-1α-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. 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, 16α-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). 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). 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. 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. Is E2 a Genotoxic or Epigenetic Carcinogen? 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 β 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). Conclusions 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 of synthetic and steroidal estrogens and their metabolites reported previously (3–7). Mutagenicity assays are designed to detect only the relatively high-mutation frequencies of potent carcinogenic compounds. In contrast, estrogens may be only weakly mutagenic, as is to be expected for endogenous compounds. Thus, mutagenicity assay conditions may have to be redesigned to detect low mutation frequencies at multiple gene loci with high accuracy and precision. The current data presented in this chapter lead to the conclusion that estrogens are genotoxic carcinogens. Oxidation of E1 and E2 to CE, with the 4-CE playing the major role, is the critical pathway of activation to form ultimate electrophilic metabolites and ROS. Table 1. Correlation of depurinating adducts with H-ras mutations in mouse skin papillomas*     H-ras mutations  PAH  Major DNA adducts (%)  No. of mutations/ No. of mice  Codon  *PAH = polycyclic aromatic hydrocarbons; DMBA = 7,12-dimethylbenz[a]anthracene; DB[a,l]P = dibenzo[a,l]pyrene; and BP = benzo[a]pyrene.  DMBA  N7Ade (79)  4/4 CAA → CTA  61  DB[a,l]P  N7Ade (32)  4/5 CAA → CTA  61    N3Ade (49)      BP  C8Gua + N7Gua (46)  10/20 GGC → GTC  13    N7Ade (25)  5/20 CAA → CTA  61       H-ras mutations  PAH  Major DNA adducts (%)  No. of mutations/ No. of mice  Codon  *PAH = polycyclic aromatic hydrocarbons; DMBA = 7,12-dimethylbenz[a]anthracene; DB[a,l]P = dibenzo[a,l]pyrene; and BP = benzo[a]pyrene.  DMBA  N7Ade (79)  4/4 CAA → CTA  61  DB[a,l]P  N7Ade (32)  4/5 CAA → CTA  61    N3Ade (49)      BP  C8Gua + N7Gua (46)  10/20 GGC → GTC  13    N7Ade (25)  5/20 CAA → CTA  61   View Large Table 2. Frequency of the Harvey-ras mutation in codon 61 (CAA → CTA) after treatment of mouse skin with dibenzo[a,l]pyrene Time after treatment, days  % of H-ras genes with codon 61 mutations  *Data taken from (25).  0  <0.001  1  0.1  2  1  3  5  6  0.5  9  <0.001  35  0.1  63 (tumors)  14–47   Time after treatment, days  % of H-ras genes with codon 61 mutations  *Data taken from (25).  0  <0.001  1  0.1  2  1  3  5  6  0.5  9  <0.001  35  0.1  63 (tumors)  14–47   View Large Table 3. Reaction of E2−3,4-Q and HRP-, LP-, or P450-activated 4-OHE2 with DNA*   Depurinating adducts, μmol/mol DNA-P      Compound  4-OHE2−1(α,β)-N7Gua  4-OHE2−1(α,β)-N3Ade  Stable adducts, μmol/mol DNA-P  Ratio of depurinating to stable adducts  *HRP = horseradish peroxidase; LP = lactoperoxidase; P450 = cytochrome P450 in phenobarbitol-induced rat liver microsomes; ND = not yet determined.  E2−3,4-Q  186  196  0.06  6367  4-OHE2          + HRP  197  198  0.03  13 167  + LP  363  208  0.05  11 420  + P450  125  133  ND       Depurinating adducts, μmol/mol DNA-P      Compound  4-OHE2−1(α,β)-N7Gua  4-OHE2−1(α,β)-N3Ade  Stable adducts, μmol/mol DNA-P  Ratio of depurinating to stable adducts  *HRP = horseradish peroxidase; LP = lactoperoxidase; P450 = cytochrome P450 in phenobarbitol-induced rat liver microsomes; ND = not yet determined.  E2−3,4-Q  186  196  0.06  6367  4-OHE2          + HRP  197  198  0.03  13 167  + LP  363  208  0.05  11 420  + P450  125  133  ND     View Large Table 4. Testosterone and estradiol-induced changes in dorsolateral prostate*   Periurethral  Periphery  Endpoint measured  Control  Treated  % Change  Control  Treated  % Change  *From (67) and M. Bosland; unpublished data.  †P = .02; ND = not detectable.  Adenocarcinoma  0  10/12  83  0  0    DNA adducts  2.7  10.2*  380  ND  ND    Lipid hydroperoxides  1.4  4.5  320  2.0  3.8  190  8-OHdG  0.3  2.1†  700  ND  0.1       Periurethral  Periphery  Endpoint measured  Control  Treated  % Change  Control  Treated  % Change  *From (67) and M. Bosland; unpublished data.  †P = .02; ND = not detectable.  Adenocarcinoma  0  10/12  83  0  0    DNA adducts  2.7  10.2*  380  ND  ND    Lipid hydroperoxides  1.4  4.5  320  2.0  3.8  190  8-OHdG  0.3  2.1†  700  ND  0.1     View Large Table 5. Effects of estradiol (E2) on immune responses* *IL = interleukin; Ig = immunoglobulin.  E2 induces IL-1α  E2 decreases IL-1α-induced IL-6  • Chemotactic factors (IL-8, LTB4)  • Human epithelial cell proliferation  • Infiltration of phagocytic cells  • Differentiation of T and B cells  • Immunomodulation (IL-6)  • Induction of aromatase  E2 induces aromatase in macrophages  E2 inhibits natural killer activity  E2 increases immunoglobulins (IgM, IgG, etc.)   *IL = interleukin; Ig = immunoglobulin.  E2 induces IL-1α  E2 decreases IL-1α-induced IL-6  • Chemotactic factors (IL-8, LTB4)  • Human epithelial cell proliferation  • Infiltration of phagocytic cells  • Differentiation of T and B cells  • Immunomodulation (IL-6)  • Induction of aromatase  E2 induces aromatase in macrophages  E2 inhibits natural killer activity  E2 increases immunoglobulins (IgM, IgG, etc.)   View Large Table 6. Selected properties of estrogens Carcinogenesis susceptible organs  Oxidative estrogen metabolism  Oxidant formation and lipid peroxidation  Genetic damage  Genetic factors  • DNA adducts  • Immediate early gene (c-fos, c-jun)  • Oxidized DNA bases  • Transcription factors (AP-1)  • Lipid-derived aldehyde– DNA adducts  • Growth factors (TGF-β)    • Oncogenes (c-myc, bcl-2)  Epithelial cell growth ( ↑) and cell differentiation (↓)  Increase of macrophage-mediated immune responses  • Proliferation  • Activation  • Secretions(cytokines, ROS, RNS)  • Function (Antigen processing and antigen presenting to T cells)  Carcinogenesis susceptible organs  Oxidative estrogen metabolism  Oxidant formation and lipid peroxidation  Genetic damage  Genetic factors  • DNA adducts  • Immediate early gene (c-fos, c-jun)  • Oxidized DNA bases  • Transcription factors (AP-1)  • Lipid-derived aldehyde– DNA adducts  • Growth factors (TGF-β)    • Oncogenes (c-myc, bcl-2)  Epithelial cell growth ( ↑) and cell differentiation (↓)  Increase of macrophage-mediated immune responses  • Proliferation  • Activation  • Secretions(cytokines, ROS, RNS)  • Function (Antigen processing and antigen presenting to T cells)  View Large Table 7. Effects of tamoxifen, an antitumor promoter Suppresses hyperplasia  Antagonizes inflammatory responses  Inhibits oxidant formation by neutrophils  Decreases oxidative DNA base damage  Decreases lipid peroxidation  Reduces MDA serum levels in breast cancer patients  Enhances apoptosis of tumor cells  Suppresses hyperplasia  Antagonizes inflammatory responses  Inhibits oxidant formation by neutrophils  Decreases oxidative DNA base damage  Decreases lipid peroxidation  Reduces MDA serum levels in breast cancer patients  Enhances apoptosis of tumor cells  View Large Fig. 1. View largeDownload slide Metabolic activation of DB[a,l]P by the diol epoxide and radical cation pathways. Fig. 1. View largeDownload slide Metabolic activation of DB[a,l]P by the diol epoxide and radical cation pathways. Fig. 2. View largeDownload slide Formation of stable and depurinating DNA adducts and generation of apurinic sites. Fig. 2. View largeDownload slide Formation of stable and depurinating DNA adducts and generation of apurinic sites. Fig. 3. View largeDownload slide Structures of three potent carcinogenic polycyclic aromatic hydrocarbon. Fig. 3. View largeDownload slide Structures of three potent carcinogenic polycyclic aromatic hydrocarbon. Fig. 4. View largeDownload slide Determination of DNA adducts and Harvey-ras mutations in mouse skin. Fig. 4. View largeDownload slide Determination of DNA adducts and Harvey-ras mutations in mouse skin. Fig. 5. View largeDownload slide Mouse Harvey-ras mutations. The normal Harvey-ras proto-oncogene (top) can be activated by mutation at codon 61 (CAA → CTA, middle) or codon 13 (GGC → GTC, bottom). Fig. 5. View largeDownload slide Mouse Harvey-ras mutations. The normal Harvey-ras proto-oncogene (top) can be activated by mutation at codon 61 (CAA → CTA, middle) or codon 13 (GGC → GTC, bottom). Fig. 6. View largeDownload slide Possible scheme for inducing A → T mutations from depurinating Ade adducts. Fig. 6. View largeDownload slide Possible scheme for inducing A → T mutations from depurinating Ade adducts. Fig. 7. View largeDownload slide Activating and deactivating (protecting) pathways of estrogen metabolism and formation of DNA adducts. Fig. 7. View largeDownload slide Activating and deactivating (protecting) pathways of estrogen metabolism and formation of DNA adducts. Fig. 8. View largeDownload slide Reaction of E1(E2)-3,4-Q with dG or Ade. Fig. 8. View largeDownload slide Reaction of E1(E2)-3,4-Q with dG or Ade. Fig. 9. View largeDownload slide Reaction of E1(E2)-2,3-Q with dG or dA. Fig. 9. View largeDownload slide Reaction of E1(E2)-2,3-Q with dG or dA. Fig. 10. View largeDownload slide Methodology of in vitro binding of CE-Q and activated CE to DNA. Fig. 10. View largeDownload slide Methodology of in vitro binding of CE-Q and activated CE to DNA. Fig. 11. View largeDownload slide Examples of oxidized DNA bases. Fig. 11. View largeDownload slide Examples of oxidized DNA bases. Fig. 12. View largeDownload slide Formation of HmdU and 8-OHdG in 7,12-dimethylbenz[a]anthracene (DMBA)-treated SENCAR mouse skin DNA. Adapted from (68). Fig. 12. View largeDownload slide Formation of HmdU and 8-OHdG in 7,12-dimethylbenz[a]anthracene (DMBA)-treated SENCAR mouse skin DNA. Adapted from (68). Fig. 13. View largeDownload slide Oxidative metabolism of E2. Adapted from (44) and (87). Fig. 13. View largeDownload slide Oxidative metabolism of E2. Adapted from (44) and (87). Fig. 14. View largeDownload slide Redox cycling of catechols. Fig. 14. View largeDownload slide Redox cycling of catechols. Fig. 15. View largeDownload slide Futile estrogen metabolism. Adapted from (94). Fig. 15. View largeDownload slide Futile estrogen metabolism. Adapted from (94). Fig. 16. View largeDownload slide Estradiol-induced formation of lipid hydroperoxides (LPH) and DNA damage. Adapted from (66). Fig. 16. View largeDownload slide Estradiol-induced formation of lipid hydroperoxides (LPH) and DNA damage. Adapted from (66). Fig. 17. View largeDownload slide Oxygen-derived, enzyme-driven major cellular ROS at physiologic pH. Summary of literature by Khan AU, Frenkel K. Fig. 17. View largeDownload slide Oxygen-derived, enzyme-driven major cellular ROS at physiologic pH. Summary of literature by Khan AU, Frenkel K. Fig. 18. View largeDownload slide Histogram of Anti-HMdU autoantibody titers in human sera (healthy controls, those with benign breast diseases [BBD], and those who were apparently healthy at the time of blood donation but were diagnosed with breast cancer 0.5–6 years later). Adapted from (84). Fig. 18. View largeDownload slide Histogram of Anti-HMdU autoantibody titers in human sera (healthy controls, those with benign breast diseases [BBD], and those who were apparently healthy at the time of blood donation but were diagnosed with breast cancer 0.5–6 years later). Adapted from (84). Fig. 19. View largeDownload slide Interaction among estrogen, immune system, tumor, and oxidative stress. Fig. 19. View largeDownload slide Interaction among estrogen, immune system, tumor, and oxidative stress. Supported by Public Health Service grants R01CA25176, R01CA49917, and P01CA49210 (to E. Cavalieri and E. Rogan) (National Cancer Institute [NCI]), R01CA37858 (NCI) and R01AG14587 (National Institute on Aging) (to K. Frenkel), CA63129 and CA74971 (to J. Liehr) (NCI), and CA52584 (to D. Roy) (NCI), National Institutes of Health, Department of Health and Human Services. E. Cavalieri and E. Rogan thank the key contributors to this research: D. Chakravarti, P. Devanesan, K.-M. Li, D. Stack, and R. Todorovic. K. Frenkel acknowledges the contributions of M. Bosland and R. Yasuf from New York University School of Medicine (NY) for sharing their unpublished data. References 1 Overall evaluations of carcinogenicity: an updating of IARC monographs volumes 1 to 42. IARC Monographs on the Evaluation of Carcinogenic Risk to Humans. Suppl 7. Lyon (France); 1987. p. 272–310. Google Scholar 2 Hormonal contraception and post-menopausal hormonal therapy. IARC Monographs on the Evaluation of Carcinogenic Risks to Humans. Vol 72. Lyon (France); 1999. Google Scholar 3 Lang R, Redmann U. Non-mutagenicity of some sex hormones in the Ames salmonella/microsome mutagenecity test. Mutat Res  1979; 67: 361–5. Google Scholar 4 Liehr JG, Fang WF, Sirbasku DA, Ari-Ulubelen A. Carcinogenicity of catechol estrogens in Syrian hamsters. J Steroid Biochem  1986; 24: 353–6. Google Scholar 5 Lang R, Reimann R. Studies for a genotoxic potential of some endogenous and exogenous sex steroids. I. Communication: examination for the induction of gene mutations using the Ames Salmonella/microsome test and the HGPRT test in V79 cells. Environ Mol Mutagen  1993; 21: 272–304. Google Scholar 6 Drevon C, Piccoli C, Montesano R. Mutagenicity assays of estrogenic hormones in mammalian cells. Mutat Res  1981; 89: 83–90. Google Scholar 7 Rajah TT, Pento JT. The mutagenic potential of antiestrogens at the HPRT locus in V79 cells. Res Comm Mol Pathol Pharmacol  1995; 89: 85–92. Google Scholar 8 Li JJ, Li SA. Estrogen carcinogenesis in hamster tissues: a critical review. Endocr Rev  1990; 11: 524–31. Google Scholar 9 Barrett JC, Tsutsui T. Mechanisms of estrogen-associated carcinogenesis. In: Huff J, Boyd J, Barrett JC, editors. Cellular and molecular mechanisms of hormonal carcinogenesis: environmental influences. New York (NY): Wiley-Liss; 1996. p. 105–12. Google Scholar 10 Feigelson HS, Henderson BE. Estrogens and breast cancer. Carcinogenesis  1996; 17: 2279–84. Google Scholar 11 Clarke RB, Howell A, Potten CS, Anderson E. Dissociation between steroid receptor expression and cell proliferation in the human breast. Cancer Res  1997; 57: 4987–91. Google Scholar 12 Russo J, Ao X, Grill C, Russo IH. Pattern of distribution of cells positive for estrogen receptor α and progesterone receptor in relation to proliferating cells in the mammary gland. Breast Cancer Res Treat  1999; 53: 217–27. Google Scholar 13 Endo S, Metzler M, Hieber L. Nonrandom karyotypic changes in a spontaneously immortalized and tumourigenic Syrian hamster embryo cell line. Carcinogenesis  1994; 15: 2387–90. Google Scholar 14 Liehr JG, Gladek A, Macatee T, Randerath E, Randerath K. DNA adduct formation in liver and kidney of male Syrian hamsters treated with estrogen and/or α-naphthoflavone. Carcinogenesis  1991; 12: 385–9. Google Scholar 15 Liehr JG, Wheeler WJ. Inhibition of estrogen-induced renal carcinoma in Syrian hamsters by vitamin C. Cancer Res  1983; 43: 4638–42. Google Scholar 16 Zhu BT, Roy D, Liehr JG. The carcinogenic activity of ethinyl estrogens is determined by both their hormonal characteristics and their conversion to catechol metabolites. Endocrinology  1993; 132: 577–83. Google Scholar 17 Stalford AC, Maggs JL, Gilchrist TL, Park BK. Catecholestrogens as mediators of carcinogenesis: correlation of aromatic hydroxylation of estradiol and its fluorinated analogs with tumor induction in Syrian hamsters. Mol Pharmacol  1994; 45: 1259–67. Google Scholar 18 Weinberg RA. How cancer arises. Sci Am  1996; 275: 62–77. Google Scholar 19 Cavalieri EL, Rogan EG. The approach to understanding aromatic hydrocarbon carcinogenesis. The central role of radical cations in metabolic activation. Pharmacol Ther  1992; 55: 183–99. Google Scholar 20 Cavalieri EL, Rogan EG. Mechanisms of tumor initiation by polycyclic aromatic hydrocarbons in mammals. In: Neilson AH, editor. The handbook of environmental chemistry. Vol 3J. PAHs and related compounds. Heidelberg (Germany): Springer-Verlag; 1998. p. 81–117. Google Scholar 21 Chakravarti D, Pelling JC, Cavalieri EL, Rogan EG. Relating aromatic hydrocarbon-induced DNA adducts and c-H-ras mutations in mouse skin papillomas: the role of apurinic sites. Proc Natl Acad Sci U S A  1995; 92: 10422–6. Google Scholar 22 Devanesan PD, RamaKrishna NV, Padmavathi NS, Higginbotham S, Rogan EG, Cavalieri EL, et al. Identification and quantitation of 7,12-dimethylbenz[a]anthracene–DNA adducts formed in mouse skin. Chem Res Toxicol  1993; 6: 364–71. Google Scholar 23 Chen L, Devanesan PD, Higginbotham S, Ariese F, Jankowiak R, Small GJ, et al. Expanded analysis of benzo[a]pyrene–DNA adducts formed in vitro and in mouse skin: their significance in tumor initiation. Chem Res Toxicol  1996; 9: 897–903. Google Scholar 24 Chakravarti D, Mailander P, Higginbotham S, Cavalieri E, Rogan E. Relationship of depurinating adducts with H-ras oncogenic mutations in mouse skin papillomas induced by benzo[a]pyrene, benzo[a]pyrene-7,8-dihydrodiol and 6-methylbenzo[a]pyrene. Proc Am Assoc Cancer Res  1999; 40: 507. Google Scholar 25 Chakravarti D, Mailander P, Franzen J, Higginbotham S, Cavalieri E, Rogan E. Detection of dibenzo[a,l]pyrene-induced H-ras codon 61 mutant genes in preneoplastic SENCAR mouse skin using a new PCR–RFLP method. Oncogene  1998; 16: 3203–10. Google Scholar 26 Stack DE, Byun J, Gross ML, Rogan EG, Cavalieri EL. Molecular characteristics of catechol estrogen quinones in reactions with deoxyribonucleosides. Chem Res Toxicol  1996; 9: 851–9. Google Scholar 27 Cavalieri EL, Stack DE, Devanesan PD, Todorovic R, Dwivedy I, Higginbotham S, et al. Molecular origin of cancer: catechol estrogen-3,4-quinones as endogenous tumor initiators. Proc Natl Acad Sci U S A  1997; 94: 10937–42. Google Scholar 28 Ball P, Knuppen R. Catecholoestrogens (2- and 4-hydroxyestrogens): chemistry, biogenesis, metabolism, occurrence and physiological significance. Acta Endocrinol Suppl (Copenh)  1980; 232: 1–127. Google Scholar 29 Martucci CP, Fishman J. P450 enzymes of estrogen metabolism. Pharmacol Ther  1993; 57: 237–57. Google Scholar 30 Li KM, Devanesan PD, Rogan EG, Cavalieri EL. Formation of the depurinating 4-hydroxyestradiol (4-OHE2)-1-N7Gua and 4-OHE2-1-N3Ade adducts by reaction of E2-3,4-quinone with DNA. Proc Am Assoc Cancer Res  1998; 39: 636. Google Scholar 31 Mulder PP, Chen L, Sekhar BC, George M, Gross ML, Rogan EG, et al. Synthesis and structure determination of the adducts formed by electrochemical oxidation of 1,2,3,4-Tetrahydro-7,12-dimethylbenz[a]anthracene in the presence of deoxyribonucleosides or adenine. Chem Res Toxicol  1996; 9: 1264–77. Google Scholar 32 Li KM, Todorovic R, Rogan EG, Cavalieri EL, Ariese F, Suh M, et al. Identification and quantitation of dibenzo[a,l]pyrene—DNA adducts formed by rat liver microsomes in vitro: preponderance of depurinating adducts. Biochemistry  1995; 34: 8043–9. Google Scholar 33 Weisz J, Bui QD, Roy D, Liehr JG. Elevated 4-hydroxylation of estradiol by hamster kidney microsomes: a potential pathway of metabolic activation of estrogens. Endocrinology  1992; 131: 655–61. Google Scholar 34 Zhu BT, Bui QD, Weisz J, Liehr JG. Conversion of estrone to 2- and 4-hydroxyestrone by hamster kidney and liver microsomes: implications for the mechanism of estrogen-induced carcinogenesis. Endocrinology  1994; 135: 1772–9. Google Scholar 35 Bui Q, Weisz J. Identification of microsomal, organic hydroperoxide-dependent catechol estrogen formation: comparison with NADPH-dependent mechanism. Pharmacology  1988; 36: 356–64. Google Scholar 36 Bunyagidj C, McLachlan JA. Catechol estrogen formation in mouse uterus. J Steroid Biochem  1988; 31: 795–801. Google Scholar 37 Li JJ, Li SA. Estrogen carcinogenesis in Syrian hamster tissues: role of metabolism. Fed Proc  1987; 46: 1858–63. Google Scholar 38 Liehr JG, Ricci MJ, Jefcoate CR, Hannigan EV, Hokanson JA, Zhu BT. 4-Hydroxylation of estradiol by human uterine myometrium and myoma microsomes: implications for the mechanism of uterine tumorigenesis. Proc Natl Acad Sci U S A  1995; 92: 9220–4. Google Scholar 39 Liehr JG, Ricci MJ. 4-Hydroxylation of estrogens as marker of human mammary tumors. Proc Natl Acad Sci U S A  1996; 93: 3294–6. Google Scholar 40 Castagnetta LA, Granata OM, Arcuri FP, Polito LM, Rosati F, Cartoni GP. Gas chromatography/mass spectrometry of catechol estrogens. Steroids  1992; 57: 437–43. Google Scholar 41 Spink DC, Hayes CL, Young NR, Christou M, Sutter TR, Jefcoate CR, et al. The effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin on estrogen metabolism in MCF-7 breast cancer cells: evidence for induction of a novel 17β-estradiol 4-hydroxylase. J Steroid Biochem Mol Biol  1994; 51: 251–8. Google Scholar 42 Hayes CL, Spink DC, Spink BC, Cao JQ, Walker NJ, Sutter TR. 17β-estradiol hydroxylation catalyzed by human cytochrome P450 1B1. Proc Natl Acad Sci U S A  1996; 93: 9776–81. Google Scholar 43 Spink DC, Spink BC, Cao JQ, Gierthy JF, Hayes CL, Li Y, et al. Induction of cytochrome P450 1B1 and catechol estrogen metabolism in ACHN human renal adenocarcinoma cells. J Steroid Biochem Mol Biol  1997; 62: 223–32. Google Scholar 44 Spink DC, Spink BC, Cao JQ, DePasquale JA, Pentecost BT, Fasco MJ, et al. Differential expression of CYP1A1 and CYP1B1 in human breast epithelial cells and breast tumor cells. Carcinogenesis  1998; 19: 291–8. Google Scholar 45 Wolff MS, Toniolo PG, Lee EW, Rivera M, Dubin N. Blood levels of organochlorine residues and risk of breast cancer. J Natl Cancer Inst  1993; 85: 648–52. Google Scholar 46 Li JJ, Li SA, Klicka JK, Parsons JA, Lam LK. Relative carcinogenic activity of various synthetic and natural estrogens in the Syrian hamster kidney. Cancer Res  1983; 43: 5200–4. Google Scholar 47 Kirkman H. Estrogen-induced tumors of the kidney in the Syrian hamster. III. Growth characteristics in the Syrian hamster. Monogr Natl Cancer Inst  1959; 1: 1–57. Google Scholar 48 Devanesan P, Todorovic R, Zhao J, Higginbotham S, Gross M, Rogan E, et al. Formation of catechol estrogen adducts in kidneys of male hamsters treated with 4-hydroxyestradiol. Proc Am Assoc Cancer Res  1999; 40: 46. Google Scholar 49 Cao K, Stack DE, Ramanathan R, Gross ML, Rogan ER, Cavalieri EL. Synthesis and structure elucidation of estrogen quinones conjugated with cysteine, N-acetylcysteine, and glutathione. Chem Res Toxicol  1998; 11: 909–16. Google Scholar 50 Cao K, Devanesan PD, Ramanathan R, Gross ML, Rogan ER, Cavalieri EL. Covalent binding of catechol estrogens to glutathione catalyzed by horseradish peroxidase, lactoperoxidase, or rat liver microsomes. Chem Res Toxicol  1998; 11: 917–24. Google Scholar 51 Liehr JG, Ballatore AM, Dague BB, Ulubelen AA. Carcinogenicity and metabolic activation of hexestrol. Chem Biol Interact  1985; 55: 157–76. Google Scholar 52 Haaf H, Metzler M. In vitro metabolism of diethylstilbestrol by hepatic, renal and uterine microsomes of rats and hamsters. Effects of difference inducers. Biochem Pharmacol  1985; 34: 3107–15. Google Scholar 53 Blaich G, Gottlicher M, Cikryt P, Metzler M. Effects of various inducers on diethylstilbestrol metabolism, drug-metabolizing enzyme activities and the aromatic hydrocarbon (Ah) receptor in male Syrian golden hamster liver. J Steroid Biochem  1990; 35: 201–4. Google Scholar 54 Metzler M, McLachlan JA. Oxidative metabolism of the synthetic estrogens hexestrol and dienestrol indicates reactive intermediates. Adv Exp Med Biol  1981; 136 Pt A: 829–37. Google Scholar 55 Jan ST, Devanesan PD, Stack DE, Ramanathan R, Byun J, Gross ML, et al. Metabolic activation and formation of DNA adducts of hexestrol, a synthetic nonsteroidal carcinogenic estrogen. Chem Res Toxicol  1998; 11: 412–9. Google Scholar 56 Dizdaroglu M. Chemical determination of free radical-induced damage to DNA. Free Radic Biol Med  1991; 10: 225–42. Google Scholar 57 Frenkel K. Carcinogen-mediated oxidant formation and oxidative DNA damage. Pharmacol Ther  1992; 53: 127–66. Google Scholar 58 Shirname-More L, Rossman TG, Troll W, Teebor GW, Frenkel K. Genetic effects of 5-hydroxymethyl-2′-deoxyuridine, a product of ionizing radiation. Mutat Res  1987; 178: 177–86. Google Scholar 59 Tchou J, Grollman AP. Repair of DNA containing the oxidatively-damaged base, 8-oxoguanine. Mutat Res  1993; 299: 277–87. Google Scholar 60 Feig DI, Sowers LC, Loeb LA. Reverse chemical mutagenesis: identification of the mutagenic lesions resulting from reactive oxygen species-mediated damage to DNA. Proc Natl Acad Sci U S A  1994; 91: 6609–13. Google Scholar 61 Berkner KL, Folk WR. EcoRI cleavage and methylation of DNAs containing modified pyrimidines in the recognition sequence. J Biol Chem  1977; 252: 3185–93. Google Scholar 62 Cerda S, Weitzman SA. Influence of oxygen radical injury on DNA methylation. Mutat Res  1997; 386: 141–52. Google Scholar 63 Zingg JM, Jones PA. Genetic and epigenetic aspects of DNA methylation on genome expression, evolution, mutation and carcinogenesis. Carcinogenesis  1997; 18: 869–82. Google Scholar 64 Teebor GW. Excision DNA repair. In: Vos JM, editor. DNA repair mechanisms: impact on human diseases and cancer. Austin (TX): RG Landes Co.; 1995. p. 99–123. Google Scholar 65 Demple B, Harrison L. Repair of oxidative damage to DNA: enzymology and biology. Annu Rev Biochem  1994; 63: 915–48. Google Scholar 66 Liehr JG. Hormone-associated cancer: mechanistic similarities between human breast cancer and estrogen-induced kidney carcinogenesis in hamsters. Environ Health Perspect  1997; 105 Suppl 3: 565–9. Google Scholar 67 Han X, Liehr JG, Bosland MC. Induction of a DNA adduct detectable by 32P-postlabeling in the dorsolateral prostate of NBL/Cr rats treated with estradiol-17β and testosterone. Carcinogenesis  1995; 16: 951–4. Google Scholar 68 Frenkel K, Wei L, Wei H. 7,12-dimethylbenz[a]anthracene induces oxidative DNA modification in vivo. Free Radic Biol Med  1995; 19: 373– 80. Google Scholar 69 Dipple A, Pigott MA, Bigger CA, Blake DM. 7,12-dimethylbenz[a]-anthracene—DNA binding in mouse skin: response of different mouse strains and effects of various modifiers of carcinogenesis. Carcinogenesis  1984; 5: 1087–90. Google Scholar 70 Frenkel K, Li X, Li C. Effects of interleukin (IL)-1α on 7,12-dimethylbenz[a]anthracene (DMBA)-induced carcinogenesis in SENCAR mice. Proc Am Assoc Cancer Res  1998; 39: 487. Google Scholar 71 Cutolo M, Sulli A, Seriolo B, Accardo S, Masi AT. Estrogens, the immune response and autoimmunity. Clin Exp Rheumatol  1995; 13: 217– 26. Google Scholar 72 Kupper TS. Immune and inflammatory processes in cutaneous tissues. Mechanisms and speculations [published erratum appears in J Clin Invest 1991;87:753]. J Clin Invest  1990; 86: 1783–9. Google Scholar 73 Weitzman SA, Gordon LI. Inflammation and cancer: role of phagocyte-generated oxidants in carcinogenesis. Blood  1990; 76: 655–63. Google Scholar 74 Stadnyk AW. Cytokine production by epithelial cells. FASEB J  1994; 8: 1041–7. Google Scholar 75 Djuric Z, Everett CK, Luongo DA. Toxicity, single-strand breaks, and 5-hydroxymethyl-2′-deoxyuridine formation in human breast epithelial cells treated with hydrogen peroxide. Free Radic Biol Med  1993; 14: 541–7. Google Scholar 76 Huang X, Yusaf R, Shah R, Frenkel K. Pro-oxidant effects of 7,12-dimethylbenz[a]anthracene (DMBA) on human epithelial breast cells. Proc Am Assoc Cancer Res  1998; 39: 372. Google Scholar 77 Szatrowski TP, Nathan CF. Production of large amounts of hydrogen peroxide by human tumor cells. Cancer Res  1991; 51: 794–8. Google Scholar 78 Bhimani RS, Troll W, Grunberger D, Frenkel K. Inhibition of oxidative stress in HeLa cells by chemopreventive agents. Cancer Res  1993; 53: 4528–33. Google Scholar 79 Calaf G, Russo J. Transformation of human breast epithelial cells by chemical carcinogens. Carcinogenesis  1993; 14: 483–92. Google Scholar 80 Djuric Z, Heilbrun LK, Reading BA, Boomer A, Valeriotta FA, Martino S. Effects of a low-fat diet on levels of oxidative damage to DNA in human peripheral nucleated blood cells. J Natl Cancer Inst  1991; 83: 766–9. Google Scholar 81 Djuric Z, Heilbrun LK, Simon MS, Smith D, Luongo DA, LoRusso PM, et al. Levels of 5-hydroxymethyl-2′-deoxyuridine in DNA from blood as a marker of breast cancer risk. Cancer  1996; 77: 691–6. Google Scholar 82 Malins DC, Polissar NL, Gunselman SJ. Progression of human breast cancers to the metastatic state is linked to hydroxyl radical-induced DNA damage. Proc Natl Acad Sci U S A  1996; 93: 2557–63. Google Scholar 83 Malins DC, Polissar NL, Schaefer S, Su Y, Vinson M. A unified theory of carcinogenesis based on order-disorder transitions in DNA structure as studied in the human ovary and breast. Proc Natl Acad Sci U S A  1998; 95: 7637–42. Google Scholar 84 Frenkel K, Karkoszka J, Glassman T, Dubin N, Toniolo P, Taioli E, et al. Serum autoantibodies recognizing 5-hydroxymethyl-2′-deoxyuridine, an oxidized DNA base, as biomarkers of cancer risk in women. Cancer Epidemiol Biomarkers Prev  1998; 7: 49–57. Google Scholar 85 Trush MA, Kensler TW. An overview of the relationship between oxidative stress and chemical carcinogenesis. Free Rad Biol Med  1991; 10: 201–9. Google Scholar 86 Bui QD, Weisz J. Monooxygenase mediating catecholestrogen formation by rat anterior pituitary is an estrogen-4-hydroxylase. Endocrinology  1989; 124: 1085–7. Google Scholar 87 Shou M, Korzekwa KR, Brooks EN, Krausz KW, Gonzalez FJ, Gelboin HV. Role of human hepatic cytochrome P450 1A2 and 3A4 in the metabolic activation of estrone. Carcinogenesis  1997; 18: 207–14. Google Scholar 88 Zhu BT, Conney AH. Functional role of estrogen metabolism in target cells: review and perspectives. Carcinogenesis  1998; 19: 1–27. Google Scholar 89 Nutter LM, Wu YY, Ngo EO, Sierra EE, Gutierrez PL, Abul-Hajj YJ. An o-quinone form of estrogen produces free radicals in human breast cancer cells: correlation with DNA damage. Chem Res Toxicol  1994; 7: 23–8. Google Scholar 90 Tabakovic K, Gleason WB, Ojala WH, Abul-Hajj YJ. Oxidative transformation of 2-hydroxyestrone. Stability and reactivity of 2,3-estrone quinone and its relationship to estrogen carcinogenicity. Chem Res Toxicol  1996; 9: 860–5. Google Scholar 91 Klein CB, Frenkel K, Costa M. The role of oxidative processes in metal carcinogenesis. Chem Res Toxicol  1991; 4: 592–604. Google Scholar 92 Lloyd RV, Hanna PM, Mason RP. The origin of the hydroxyl radical oxygen in the Fenton reaction. Free Radic Biol Med  1997; 22: 885–8. Google Scholar 93 Weisz J, Clawson GA, Creveling CR. Biogenesis and inactivation of catecholestrogens. Adv Pharmacol  1998; 42: 828–33. Google Scholar 94 Sipe HJ Jr, Jordan SJ, Hanna PM, Mason RP. The metabolism of 17β-estradiol by lactoperoxidase: a possible source of oxidative stress in breast cancer. Carcinogenesis  1994; 15: 2637–43. Google Scholar 95 Pero RW, Sheng Y, Olsson A, Bryngelsson C, Lund-Pero M. Hypochlorous acid/N-chloramines are naturally produced DNA repair inhibitors. Carcinogenesis  1996; 17: 13–8. Google Scholar 96 Ding H, Demple B. In vivo kinetics of a redox-regulated transcriptional switch. Proc Natl Acad Sci U S A  1997; 94: 8445–9. Google Scholar 97 Aruoma OI. Free radicals, oxidants, and antioxidants: trend towards the year 2000 and beyond. In: Aruoma OI, Halliwell B, editors. Molecular biology of free radicals in human diseases. London (U.K.): OICA International; 1998. p. 1–28. Google Scholar 98 Wang MY, Liehr JG. Lipid hydroperoxide-induced endogenous DNA adducts in hamsters: possible mechanism of lipid hydroperoxide-mediated carcinogenesis. Arch Biochem Biophys  1995; 316: 38–46. Google Scholar 99 Nath RG, Chen HJ, Chang L, Amin S, Ocando JE, Chung FL. Detection of 4-hydroxynonenal-derived 1,N2-6-propanodeoxyguanosine adducts in the liver DNA of untreated rat by a 32P-postlabeling method. Proc Am Assoc Cancer Res  1998; 39: 490. Google Scholar 100 Ansar Ahmed S, Dauphinee MJ, Montoya AI, Talal N. Estrogen induces normal murine CD5+ B cells to produce autoantibodies. J Immunol  1989; 142: 2647–53. Google Scholar 101 Ansel J, Perry P, Brown J, Damm D, Phan T, Hart C, et al. Cytokine modulation of keratinocyte cytokines. J Invest Dermatol  1990; 94(6 Suppl): 101S–107S. Google Scholar 102 Griffiths HR, Lunec J. Molecular aspects of free radical damage in inflammatory autoimmune pathology. In: Aruoma OI, Halliwell B, editors. Molecular biology of free radicals in human diseases. London (U.K.): OICA International; 1998. p. 327–66. Google Scholar 103 Rosen GM, Pou S, Ramos CL, Cohen MS, Britigan BE. Free radicals and phagocytic cells. FASEB J  1995; 9: 200–9. Google Scholar 104 Tabibzadeh SS, Santhanam U, Sehgal PB, May LT. Cytokine-induced production of IFN-β2/IL-6 by freshly explanted human endometrial stromal cells. Modulation by estradiol-17β. J Immunol  1989; 142: 3134–9. Google Scholar 105 Mor G, Yue W, Santen RJ, Gutierrez L, Eliza M, Berstein LM, et al. Macrophages, estrogen, and the microenvironment of breast cancer. J Steroid Biochem Mol Biol  1998; 67: 403–11. Google Scholar 106 Lewis CE, Leek R, Harris A, McGee JO. Cytokine regulation of angiogenesis in breast cancer: the role of tumor-associated macrophages. J Leukoc Biol  1995; 57: 747–51. Google Scholar 107 Santner SJ, Pauley RJ, Tait L, Kaseta J, Santen RJ. Aromatase activity and expression in breast cancer and benign breast tissue stromal cells. J Clin Endocrinol Metab  1997; 82: 200–8. Google Scholar 108 Reed MJ, Topping L, Coldham NG, Purohit A, Ghilchik MW, James VH. Control of aromatase activity in breast cancer cells: the role of cytokines and growth factors. J Steroid Biochem Mol Biol  1993; 44: 589–96. Google Scholar 109 Yue W, Wang JP, Hamilton CJ, Demers LM, Santen RJ. In situ aromatization enhances breast tumor estradiol levels and cellular proliferation. Cancer Res  1998; 58: 927–32. Google Scholar 110 Esumi H, Tannenbaum SR. U.S.–Japan Cooperative Research Program: seminar on nitric oxide synthase and carcinogenesis. Cancer Res  1994; 54: 297–301. Google Scholar 111 Floyd RA. The role of 8-hydroxyguanine in carcinogenesis. Carcinogenesis  1990; 11: 1447–50. Google Scholar 112 Frenkel K. Carcinogenesis: the role of active oxygen species. In: Bertino JR, editor. Encyclopedia of cancer. San Diego (CA): Academic Press; 1997. p. 233–45. Google Scholar 113 Klein CB, Snow ET, Frenkel K. Molecular mechanisms in metal carcinogenesis: role of oxidative stress. In: Aruoma OI, Halliwell B, editors. Molecular biology of free radicals in human diseases. London (U.K.): OICA International; 1998. p. 79–137. Google Scholar 114 Jansson G. Oestrogen-induced enhancement of myeloperoxidase activity in human polymorphonuclear leukocytes—a possible cause of oxidative stress in inflammatory cells. Free Radic Res Commun  1991; 14: 195–208. Google Scholar 115 Carlsten H, Holmdahl R, Tarkowski A, Nilsson LA. Oestradiol- and testosterone-mediated effects on the immune system in normal and autoimmune mice are genetically linked and inherited as dominant traits. Immunology  1989; 68: 209–14. Google Scholar 116 Carlsten H, Verdrengh M, Taube M. Additive effects of suboptimal doses of estrogen and cortisone on the suppression of T lymphocyte dependent inflammatory responses in mice. Inflamm Res  1996; 45: 26–30. Google Scholar 117 Cooke MS, Mistry N, Wood C, Herbert KE, Lunec J. Immunogenicity of DNA damaged by reactive oxygen species—implications for anti-DNA antibodies in lupus. Free Radic Biol Med  1997; 22: 151–9. Google Scholar 118 Malins DC, Polissar NL, Nishikida K, Holmes EH, Gardner HS, Gunselman SJ. The etiology and prediction of breast cancer. Fourier transform-infrared spectroscopy reveals progressive alterations in breast DNA leading to a cancer-like phenotype in a high proportion of normal women. Cancer  1995; 75: 503–17. Google Scholar 119 Arrigo AP, Kretz-Remy C. Regulation of mammalian gene expression by free radicals. In: Aruoma OI, Halliwell B, editors. Molecular biology of free radicals in human diseases. London (U.K.): OICA International; 1998. p. 183–223. Google Scholar 120 Suzuki K, Hei TK. Induction of heme oxygenase in mammalian cells by mineral fibers: distinctive effect of reactive oxygen species. Carcinogenesis  1996; 17: 661–7. Google Scholar 121 O'Regan RM, Cisneros A, England GM, MacGregor JI, Muenzner HD, Assikis VJ, et al. Effects of the antiestrogens tamoxifen, toremifene, and ICI 182,780 on endometrial cancer growth. J Natl Cancer Inst  1998; 90: 1552–8. Google Scholar 122 Wiseman H. Tamoxifen: Molecular basis of use in cancer treatment and prevention. Chichester (U.K.): John Wiley & Sons; 1994. p. 209. Google Scholar 123 Jordan VC, editor. Long-term tamoxifen treatment for breast cancer. Madison (WI): The University of Wisconsin Press; 1994. p. 289. Google Scholar 124 Wei H, Frenkel K. Relationship of oxidative events and DNA oxidation in SENCAR mice to in vivo promoting activity of phorbol ester-type tumor promoters. Carcinogenesis  1993; 14: 1195–201. Google Scholar 125 Hertz R. The estrogen problem—retrospect and prospect. In: McLachlan JA, editor. Estrogens in the environment. II. Influences on development. New York (NY): Elsevier; 1985. p. 1–11. Google Scholar 126 Roy D, Palangat M, Chen CW, Thomas RT, Colerangle JC, Atkinson A, et al. Biochemical and molecular changes at the cellular levels in response to exposure of environmental estrogen-like chemicals. J Toxicol Environ Health  1997; 50: 1–29. Google Scholar 127 Lengauer C, Kinzler KW, Vogelstein B. Genetic instabilities in human cancers. Nature  1998; 396: 643–9. Google Scholar 128 Barrett JC, Wong A, McLachlan JA. Diethylstilbestrol induces neoplastic transformation without measurable gene mutation at two loci. Science  1981; 212: 1402–4. Google Scholar 129 Roy D, Colerangle JC, Singh KP. Is exposure of environmental or industrial endocrine disrupting estrogen-like chemicals able to cause genomic instability? Frontiers Bioscience  1998; 3: d913–21. Google Scholar 130 Roy D, Liehr JG. Estrogen. DNA damage and mutations. Mutat Res  1999; 424: 107–15. Google Scholar 131 Metzler M, Pfeiffer E, Schuler M, Rosenberg B. Effects of estrogen on microtubule assembly: significance for aneuploidy. In: Li JJ, Nandi S, Li SA, Gustafsson J, Sekely L, editors. Hormonal carcinogenesis II. New York (NY): Springer-Verlag; 1996. p. 193–9. Google Scholar 132 Metzler M, Pfeiffer E, Kohl W, Schnitzler R. Interactions of carcinogenic estrogens with microtubular proteins. In: Li JJ, Nandi S, Li SA, editors. Hormonal carcinogenesis. New York (NY): Springer-Verlag; 1992. p. 86–94. Google Scholar 133 Jones LA, Hajek RA. Effects of estrogenic chemicals on development. Environ Health Perspect  1995; 103: 63–7. Google Scholar 134 Hajek RA, Pathak S, Boddie AK, Jones LA. Aneuploidy of mouse cervicovaginal epithelium induced by prenatal estrogen treatment. Proc Am Assoc Cancer Res  1989; 30: 299. Google Scholar 135 Banerjee SH, Banerjee S, Li SA, Li JJ. Cytogenetic changes in renal neoplasms and during estrogen-induced carcinogenesis. In: Li JJ, Nandi S, Li SA, editors. Hormonal carcinogenesis. New York (NY): Springer-Verlag; 1992. p. 247–50. Google Scholar 136 Banerjee SK, Banerjee S, Li SA, Li JJ. Induction of chromosome aberrations in Syrian hamster renal cortical cells by various estrogens. Mutat Res  1994; 311: 191–7. Google Scholar 137 Tsutsui T, Suzuki N, Maizumi H, Barrett JC. Aneuploidy induction in human fibroblasts: comparison with results in Syrian hamster fibroblasts. Mutat Res  1990; 240: 241–9. Google Scholar 138 Korah RM, Humayun MZ. Mutagenic and recombinagenic effects of diethylstilbestrol quinone. Mutat Res  1993; 289: 205–14. Google Scholar 139 Roy D, Abul-Hajj YJ. Estrogen-nucleic acid adducts: guanine is major site for interaction between 3,4-estrone quinone and COIII gene. Carcinogenesis  1997; 18: 1247–9. Google Scholar 140 Yan ZJ, Roy D. Mutations of DNA polymerase β mRNA of stilbene estrogen-induced kidney tumors in Syrian hamster. Biochem Mol Biol Int  1995; 37: 175–83. Google Scholar 141 Hodgson AV, Ayala-Torres S, Thompson EB, Liehr JG. Estrogen-induced microsatellite DNA alterations are associated with Syrian hamster kidney tumorigenesis. Carcinogenesis  1998; 19: 2169–72. Google Scholar 142 Hodgson AV, Warnke PH, Liehr JG. Estrogen-induced microsatellite DNA instability during Syrian hamster kidney tumorigenesis. Proc Am Assoc Cancer Res  1999; 40: 510. Google Scholar 143 Paquette B. Enhancement of genomic instability by 17β-estradiol in minisatellite sequences of X-ray-transformed mouse 10T1/2 cells. Carcinogenesis  1996; 17: 1221–5. Google Scholar 144 Boyd J, Takahashi H, Waggoner SE, Jones LA, Hajek RA, Wharton JT, et al. Molecular genetic analysis of clear cell adenocarcinomas of the vagina and cervix associated and unassociated with diethylstilbestrol exposure in utero. Cancer  1996; 77: 507–13. Google Scholar 145 De Benedetti VM, Welsh JA, Yu MC, Bennett WP. p53 mutations in hepatocellular carcinoma related to oral contraceptive use. Carcinogenesis  1996; 17: 145–9. Google Scholar 146 Tsutsui T, Barrett JC. Neoplastic transformation of cultured mammalian cells by estrogens and estrogen-like chemicals. Environ Health Perspect  1997; 105 Supp 3: 619–24. Google Scholar 147 Jackson AL, Chen R, Loeb LA. Induction of microsatellite instability by oxidative DNA damage. Proc Natl Acad Sci U S A  1998; 95: 12468–73. Google Scholar © Oxford University Press
TI - Chapter 4: Estrogens as Endogenous Genotoxic Agents—DNA Adducts and Mutations
JO - JNCI Monographs
DO - 10.1093/oxfordjournals.jncimonographs.a024247
DA - 2000-07-01
UR - https://www.deepdyve.com/lp/oxford-university-press/chapter-4-estrogens-as-endogenous-genotoxic-agents-dna-adducts-and-k1cSN0HQs3
SP - 75
EP - 94
VL - 2000
IS - 27
DP - DeepDyve
ER -