Oxidative Metabolism of Estrogens in Cancer ... - ACS Publications

Chapter 2

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Oxidative Metabolism of Estrogens in Cancer Initiation and Prevention Eleanor G. Rogan1,2,* and Ercole L. Cavalieri1,2 1Department

of Environmental, Agricultural and Occupational Health, College of Public Health, University of Nebraska Medical Center, 984388 Nebraska Medical Center, Omaha, Nebraska 68198-4388 2Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, 986805 Nebraska Medical Center, Omaha, Nebraska 68198-6805 *E-mail: [email protected]

Oxidative metabolism of the estrogens estrone (E1) and estradiol (E2) is the critical event in the initiation of cancer by estrogens. E1 and E2 are oxidized by cytochrome P450 (CYP) to the catechol estrogens 2-OHE1(E2) and 4-OHE1(E2) and then to the catechol estrogen quinones, which react with DNA to form estrogen-DNA adducts. The E1(E2)-3,4-quinones [E1(E2)-3,4-Q] react predominantly with DNA to form the depurinating adducts 4-OHE1(E2)-1-N3Ade and 4-OHE1(E2)-1-N7Gua. Loss of these adducts forms apurinic sites in the DNA that can generate mutations leading to the initiation of cancer. When estrogen metabolism becomes unbalanced toward oxidation, larger amounts of adducts are formed, and the risk of initiating cancer is greater. Women at high risk of developing breast cancer, or diagnosed with the disease, have higher levels of estrogen-DNA adducts than women at normal risk. With unbalanced estrogen metabolism, women are six-times more likely to be diagnosed with ovarian cancer. These results and others in humans and cell culture indicate that unbalanced oxidative metabolism of estrogens with formation of estrogen-DNA adducts is a critical event in the initiation of cancer. Two compounds, N-acetylcysteine and resveratrol, efficiently block formation of estrogen-DNA adducts and, thus, are promising agents to prevent cancer. © 2015 American Chemical Society In Oxidative Stress: Diagnostics, Prevention, and Therapy Volume 2; Hepel, Maria, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.


A large body of evidence for oxidative metabolism of estrogens as a mechanism of carcinogenesis has been derived from experiments on estrogen metabolism, formation of DNA adducts, mutagenicity, cell transformation and carcinogenicity (1–3). In fact, unbalanced oxidative metabolism of the natural estrogens estrone (E1) and estradiol (E2) has been shown to be the factor that renders the estrogens weak carcinogenic compounds. The predominant pathway that leads to the initiation of cancer is formation of E1(E2)-3,4-quinones [E1(E2)-3,4-Q] and reaction of these electrophilic compounds with DNA to form the depurinating 4-OHE1(E2)-1-N3Ade and 4-OHE1(E2)-1-N7Gua adducts (Figure 1) (1–3). Error-prone repair of the resulting apurinic sites leads to mutations that can initiate cancer (4, 5).

Figure 1. Major metabolic pathway in cancer initiation by estrogens. (Reproduced with permission from reference (2). Copyright 2011 Pergamon.)

Evidence for Genotoxicity Pathway in Estrogen Carcinogenesis The most direct evidence for this pathway of genotoxicity leading to cancer initiation can be summarized as follows. Evidence that depurinating DNA adducts play a major role in cancer initiation derives from the correlation between the levels of depurinating aromatic hydrocarbon-DNA adducts and oncogenic Harvey (H)ras mutations in mouse skin papillomas (1–3). A similar correlation between the sites of formation of depurinating DNA adducts and H-ras mutations was observed in mouse skin and rat mammary gland treated with E2-3,4-Q (4, 5). Studies with cultured breast epithelial cells from humans or mice have provided evidence that initiation of cancer occurs by formation of estrogen-DNA adducts. The MCF-10F cell line is an immortalized non-transformed estrogen receptor-α (ER-α)-negative human cell line. When these cells are treated with E2 or 4-OHE2, the depurinating estrogen-DNA adducts are formed (6–9). Treatment 36 In Oxidative Stress: Diagnostics, Prevention, and Therapy Volume 2; Hepel, Maria, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.


with E2 or 4-OHE2 at doses of 0.007-3.5 nM produces transformation of these cells as detected by their ability to form colonies in soft agar (6, 9–11). The presence of the antiestrogen tamoxifen or ICI-182,780 does not prevent this transformation (10). These changes are induced to a much smaller extent by 2-OHE2. These results indicate that transformation is determined by genotoxic effects of estrogens. When estrogen-transformed MCF-10F cells, which were selected by their invasiveness, were implanted into severely compromised immune-deficient mice, the cells induced tumors (12). These results demonstrate that human breast epithelial cells lacking ER-α are transformed by the genotoxic effects of estrogen metabolites. Thus, these results support the hypothesis that formation of depurinating estrogen-DNA adducts is the critical event in the initiation of cancer by estrogens. Similarly, the immortalized, normal mouse mammary cell line E6 also forms depurinating estrogen-DNA adducts and is transformed to grow in soft agar by a single treatment with 4-OHE2 or E2-3,4-Q (13). These results demonstrate that transformation of breast cells by estrogen genotoxicity occurs in both humans and animals. Studies of transgenic mice with ER-α knocked out, ERKO/wnt-1 mice, provide further important evidence demonstrating the role of estrogen genotoxicity in the initiation of cancer. Despite the absence of ER-α, mammary tumors develop in 100% of female ERKO/wnt-1 mice, driven by the wnt-1 transgene (14, 15). The protective methoxyestrogen conjugates were not found in the mammary tissue of female ERKO/wnt-1 mice, but 4-OHE1(E2) and estrogen-glutathione (GSH) conjugates, which are formed by the catechol estrogen quinones, were detected (16). These results indicate that estrogen metabolism in these mice is unbalanced toward an excess of activating pathways and limited protective pathways. When the mice were implanted with E2 following ovariectomy at 15 days of age to remove their major source of estrogens, the E2-treated mice developed mammary tumors in a dose-dependent manner (17, 18). The mammary tumors developed even in the presence of the implanted anti-estrogen ICI-182,780 (19). These results provide strong evidence for the critical role of estrogen genotoxicity in tumor initiation. The “mainstream” proposed pathway for estrogen carcinogenesis is that ERα-mediated events increase the rate of cell proliferation, giving cells less time to repair random mutations induced by unknown causes. This unproven hypothesis is belied by a variety of evidence, most directly by the difference in carcinogenicity of the 2- and 4- catechol estrogen metabolites. The catechol estrogens 4-OHE1(E2) and 2-OHE1(E2) were tested for carcinogenic activity by subcutaneous implantation into male Syrian golden hamsters. The 4-OHE1(E2) were carcinogenic, while the 2-OHE1(E2) were not (20, 21). The two catechol estrogens were also tested in CD-1 mice by injection of the compound into newborns. Once again, the 4-catechol estrogen induced uterine adenocarcinomas, whereas the 2-catechol estrogen was borderline active (22). These results are consistent with the structure of the two catechols. The 4-catechol estrogens, when oxidized to their quinones, produce an electrophilic species that reacts very strongly with DNA by protonated 1,4-Michael addition (23), whereas the catechol estrogen-2,3-quinones react with DNA by 1,6-Michael 37 In Oxidative Stress: Diagnostics, Prevention, and Therapy Volume 2; Hepel, Maria, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

addition via an intermediate quinone methide (24). For this reason, E1(E2)-3,4-Q react with DNA to a much greater extent than the E1(E2)-2,3-Q (25), forming 97% of the depurinating adducts found in humans (26). In summary, evidence from studies of carcinogenesis in animal models and malignant transformation of human and mouse mammary cells supports the hypothesis that estrogens initiate cancer by a genotoxic mechanism.


Imbalances in Estrogen Metabolism Initiation of cancer by estrogens occurs when a relatively large amount of E1(E2)-3,4-Q reacts with DNA, contributing about 97% of the adducts. A large amount of quinone reacting with DNA is due to oxidative stress, namely, the oxidative events that give rise to the catechol quinones in great abundance. These oxidative events start with the formation of the estrogens E1 and E2 from androgens, catalyzed by CYP19 (aromatase) (Figure 2). When this enzyme is over-expressed, a large amount of estrogen is produced. The estrogens are metabolized via two major pathways: 16α-hydroxylation (not shown in Figure 2) and formation of the catechol estrogens, 2-OHE1(E2) and 4-OHE1(E2). The formation of 4-OHE1(E2), catalyzed by CYP1B1, is of critical importance. The catechol estrogens are oxidized through estrogen semiquinones to the reactive catechol estrogen quinones. Molecular oxygen can oxidize the semiquinones to quinones (Figure 2). In turn, the estrogen quinones can be reduced to semiquinones by CYP reductase. This reaction completes the redox cycle. In this process, the molecular oxygen is reduced to superoxide anion radical, which is converted to H2O2. In the presence of Fe2+, H2O2 yields the reactive hydroxyl radicals. Formation of lipid hydroperoxides can occur as the first damage by hydroxyl radicals. The lipid hydroperoxides can act as unregulated cofactors of cytochrome P450; this lack of regulation can generate an abnormal increase in the oxidation of catechol estrogens to quinones. Thus, efficient redox cycling can generate abundant catechol estrogen quinones, the ultimate carcinogenic metabolites of estrogens. Conjugation of the catechols to form glucuronides, sulfates or methoxyestrogens is very abundant in the liver, but in extrahepatic tissues the major conjugation is formation of methoxyestrogens, catalyzed by the protective enzyme catechol-O-methyltransferase (COMT). If the activity of COMT is insufficient, the oxidation of catechols to semiquinones and quinones becomes competitive (Figure 2). The quinones, E1(E2)-2,3-Q and E1(E2)-3,4-Q, can be conjugated with GSH or reduced back to catechols by the enzyme quinone reductase (NQO1 and NQO2) (27, 28). Once again, if these two protective events are insufficient, the quinones can react with DNA (Figure 2). A relatively large amount of the depurinating 4-OHE1(E2)-1-N3Ade and 4-OHE1(E2)-1 N7Gua adducts indicates unbalanced estrogen metabolism. This occurs only when the oxidative events overcome the protective events. Inhibition of adduct formation can be achieved by increased activity of the protective enzymes COMT and NQO1 and/or NQO2, or decreased activity of the oxidative enzymes CYP19 and CYP1B1. 38 In Oxidative Stress: Diagnostics, Prevention, and Therapy Volume 2; Hepel, Maria, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.


Figure 2. Formation, metabolism and DNA adducts of estrogens. Activating enzymes and depurinating DNA adducts are in red and protective enzymes are in Green. N-acetylcysteine (NAcCys, shown in blue) and resveratrol (Resv, burgundy) indicate the various points where NAcCys and Resv could improve the balance of estrogen metabolism and minimize formation of depurinating estrogen-DNA adducts. (Reproduced with permission from reference (2). Copyright 2011 Pergamon.) (see color insert)

Levels of Estrogen-DNA Adducts in Humans with and without Cancer While most of us metabolize estrogens to products that are easily excreted from the body, people at risk for cancer metabolize estrogens to increased levels of E1(E2)-3,4-Q, which can react with DNA to form the depurinating adducts 4-OHE1(E2)-1-N3Ade and 4-OHE1(E2)-1 N7Gua. These adducts are shed from DNA, and the resulting apurinic sites can be unfaithfully repaired to generate mutations leading to cancer (1–5). After the depurinating adducts are released from DNA, they travel out of cells and tissues into the bloodstream and are excreted in urine. Thus, they can be identified and quantified as biomarkers of risk of developing cancer (26, 29–34). Higher levels of depurinating estrogen-DNA adducts have been detected in analyses of urine or serum from women and men who have been diagnosed with cancer, compared to healthy controls who have never had cancer: breast, ovarian and thyroid cancer in women (26, 29, 32–34) and prostate cancer and non-Hodgkin lymphoma in men (30, 31). 39 In Oxidative Stress: Diagnostics, Prevention, and Therapy Volume 2; Hepel, Maria, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

Breast Cancer


In addition to women diagnosed with breast cancer, women that are at high risk for breast cancer have higher levels of these adducts (Figure 3) (26, 29, 32). In the largest of three such studies (32) (approximately 80 women per group), a serum sample was obtained from women at normal or high risk for breast cancer (Gail Model score >1.66% (35) and women diagnosed with breast cancer. After partial purification of an aliquot by solid phase extraction, each sample was analyzed for 40 estrogen metabolites, conjugates and depurinating DNA adducts by using ultraperformance liquid chromatography/tandem mass spectrometry (UPLC-MS/ MS).

Figure 3. Ratio of depurinating estrogen-DNA adducts to estrogen metabolites and conjugates in serum of healthy women, high-risk women and women with breast cancer. (Reproduced with permission from reference (32). Copyright 2012 Pergamon.) The risk of developing breast cancer was measured as the ratio of depurinating estrogen-DNA adducts to their respective estrogen metabolites and conjugates (Figure 3) because this ratio indicates the degree of imbalance in a person’s estrogen metabolism. The DNA adducts formed by E1(E2)-3,4-Q are predominant (97%) in this ratio, whereas the adducts formed by E1(E2)-2,3-Q are minimal (3%) 40 In Oxidative Stress: Diagnostics, Prevention, and Therapy Volume 2; Hepel, Maria, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.


(26, 29, 32). The typically low ratio in women at normal risk for breast cancer indicates that their estrogen metabolism is balanced and they form relatively few estrogen-DNA adducts. Subject characteristics did not affect the highly significant differences observed between the normal risk women and the women at high risk for breast cancer or diagnosed with it. Thus, these studies demonstrate that unbalanced estrogen metabolism leading to increased levels of estrogen-DNA adducts is associated with high risk of developing breast cancer. This study (32), as well as the other two studies of women with and without breast cancer (26, 29), provide strong evidence that formation of estrogen-DNA adducts is a critical factor in the etiology of breast cancer. Thyroid Cancer Well-differentiated thyroid cancer most frequently occurs in premenopausal women, and greater exposure to estrogens may be a risk factor for this type of cancer. To investigate the role of estrogens in thyroid cancer, a spot urine sample was obtained from 40 women with thyroid cancer and 40 age-matched controls (33). Thirty-eight estrogen metabolites, conjugates and DNA adducts were analyzed by using UPLC-MS/MS, and the ratio of adducts to metabolites and conjugates was calculated for each sample. The ratio of depurinating estrogen-DNA adducts to estrogen metabolites and conjugates significantly differed between cases and controls (p

Oxidative Metabolism of Estrogens in Cancer ... - ACS Publications

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