Estrogen DNA Adducts Abstracts 1

By Ronald Steriti, ND, PhD
©

Potential mechanisms of estrogen quinone carcinogenesis.
            (Bolton and Thatcher, 2008) Download
There is a clear association between the excessive exposure to estrogens and the development of cancer in hormone-sensitive tissues (breast, endometrium). It has become clear that there are likely multiple overlapping mechanisms of estrogen carcinogenesis. One major pathway is the extensively studied hormonal pathway, by which estrogen stimulates cell proliferation through nuclear estrogen receptor (ER)-mediated signaling, thus resulting in an increased risk of genomic mutations during DNA replication. A similar "nongenomic pathway", potentially involving newly discovered membrane-associated ERs, also appears to regulate extranuclear estrogen signaling pathways. This perspective is focused on a third pathway involving the metabolism of estrogens to catechols mediated by cytochrome P450 and further oxidation of these catechols to estrogen o-quinones. Oxidative enzymes, metal ions, and in some cases molecular oxygen can catalyze o-quinone formation, so that these electrophilic/redox-active quinones can cause damage within cells by alkylation and/or oxidation of cellular proteins and DNA in many tissues. It appears that the endogenous estrogen quinones primarily form unstable N3-adenine or N7-guanine DNA adducts, ultimately resulting in mutagenic apurinic sites. In contrast, equine estrogen quinones, formed from estrogens present in popular hormone replacement therapy prescriptions, generate a variety of DNA lesions, including bulky stable adducts, apurinic sites, DNA strand cleavage, and oxidation of DNA bases. DNA damage induced by these equine quinones is significantly increased in cells containing ERs, leading us to hypothesize a mechanism involving ER binding/alkylation by the catchol/quinone, resulting in a "Trojan horse". The "Trojan horse" carries the highly redox-active catechol to estrogen -sensitive genes, where high amounts of reactive oxygen species are generated, causing selective DNA damage. Our data further suggest that other key protein targets for estrogen o-quinones could be redox-sensitive enzymes (i.e, GST P1-1, QR). These proteins are involved in stress response cascades that are known to contribute to the regulation of cell proliferation and apoptosis. Finally, it has been shown that catechol estrogens can transform breast epithelial cells into a tumorigenic phenotype and that these transformed cells had differential gene expression of several genes involved in oxidative stress. Given the direct link between excessive exposure to estrogens, metabolism of estrogens, and increased risk of breast cancer, it is crucial that factors that affect the formation, reactivity, and cellular targets of estrogen quinoids be thoroughly explored.

Catechol quinones of estrogens in the initiation of breast, prostate, and other human cancers: keynote lecture.
            (Cavalieri and Rogan, 2006) Download
Estrogens can be converted to electrophilic metabolites, particularly the catechol estrogen-3,4-quinones, estrone(estradiol)-3,4-quinone [E(1)(E(2))-3,4-Q], which react with DNA to form depurinating adducts. These adducts are released from DNA to generate apurinic sites. Error-prone repair of this damage leads to the mutations that initiate breast, prostate, and other types of cancer. The reaction of E(1)(E(2))-3,4-Q with DNA forms the depurinating adducts 4-hydroxyE(1)(E(2))-1-N3adenine [4-OHE(1)(E(2))-1-N3Ade] and 4-OHE(1)(E(2))-1-N7guanine(Gua). These two adducts constitute >99% of the total DNA adducts formed. The E(1)(E(2))-2,3-Q forms small amounts of the depurinating 2-OHE(1)(E(2))-6-N3Ade adducts. Reaction of the quinones with DNA occurs more abundantly when estrogen metabolism is unbalanced. Such an imbalance is the result of overexpression of estrogen-activating enzymes and/or deficient expression of deactivating (protective) enzymes. Excessive formation of E(1)(E(2))-3,4-Q is the result of this imbalance. Oxidation of catechols to semiquinones and quinones is a mechanism of tumor initiation not only for endogenous estrogens, but also for synthetic estrogens such as hexestrol and diethylstilbestrol, a human carcinogen. This mechanism is also involved in the initiation of leukemia by benzene, rat olfactory tumors by naphthalene, and neurodegenerative diseases such as Parkinson's disease by dopamine. In fact, dopamine quinone reacts with DNA similarly to the E(1)(E(2))-3,4-Q, forming analogous depurinating N3Ade and N7Gua adducts. The depurinating adducts that migrate from cells and can be found in body fluids can also serve as biomarkers of cancer risk. In fact, a higher level of estrogen-DNA adducts has been found in the urine of men with prostate cancer and in women with breast cancer compared to healthy controls. This unifying mechanism of the origin of cancer and other diseases suggests preventive strategies based on the level of depurinating DNA adducts that generate the first critical step in the initiation of diseases.

Depurinating estrogen-DNA adducts in the etiology and prevention of breast and other human cancers.
            (Cavalieri and Rogan, 2010) Download
Experiments on estrogen metabolism, formation of DNA adducts, mutagenicity, cell transformation and carcinogenicity have led to and supported the hypothesis that the reaction of specific estrogen metabolites, mostly the electrophilic catechol estrogen-3,4-quinones, with DNA can generate the critical mutations to initiate breast and other human cancers. Analysis of depurinating estrogen-DNA adducts in urine demonstrates that women at high risk of, or with breast cancer, have high levels of the adducts, indicating a critical role for adduct formation in breast cancer initiation. Men with prostate cancer or non-Hodgkin lymphoma also have high levels of estrogen-DNA adducts. This knowledge of the first step in cancer initiation suggests the use of specific antioxidants that can block formation of the adducts by chemical and biochemical mechanisms. Two antioxidants, N-acetylcysteine and resveratrol, are prime candidates to prevent breast and other human cancers because in various M in vitro and in vivo experiments, they reduce the formation of estrogen-DNA adducts.

The molecular etiology of breast cancer: evidence from biomarkers of risk.
            (Gaikwad et al., 2008) Download
Estrogens can become endogenous carcinogens via formation of catechol estrogen quinones, which react with DNA to form specific depurinating estrogen-DNA adducts. The mutations resulting from these adducts can lead to cell transformation and the initiation of breast cancer. Estrogen metabolites, conjugates and depurinating DNA adducts in urine samples from 46 healthy control women, 12 high-risk women and 17 women with breast cancer were analyzed. The estrogen metabolites, conjugates and depurinating DNA adducts were identified and quantified by using ultraperformance liquid chromatography/tandem mass spectrometry. The levels of the ratios of depurinating DNA adducts to their respective estrogen metabolites and conjugates were significantly higher in high-risk women (p < 0.001) and women with breast cancer (p < 0.001) than in control subjects. The high-risk and breast cancer groups were not significantly different (p = 0.62). After adjusting for patient characteristics, these ratios were still significantly associated with health status. Thus, the depurinating estrogen-DNA adducts are possible biomarkers for early detection of breast cancer risk and response to preventive treatment.

Urinary biomarkers suggest that estrogen-DNA adducts may play a role in the aetiology of non-Hodgkin lymphoma.
            (Gaikwad et al., 2009b) Download
A variety of evidence suggests that estrogens may induce non-Hodgkin lymphoma (NHL). The reaction of catechol estrogen quinones with DNA to form depurinating estrogen-DNA adducts is hypothesized to initiate this process. These adducts are released from DNA, shed from cells into the bloodstream and excreted in urine. The aim of this study was to determine whether or not the depurinating estrogen-DNA adducts might be involved in the aetiology of human NHL. Estrogen metabolites, conjugates and depurinating DNA adducts were identified and quantified in spot urine samples from 15 men with NHL and 30 healthy control men by using ultraperformance liquid chromatography/tandem mass spectrometry. The levels of estrogen-DNA adducts were significantly higher in the men with NHL than in the healthy control men. Thus, formation of estrogen-DNA adducts may play a critical role in the aetiology of NHL, and these adducts could be potential biomarkers of NHL risk.

Urine biomarkers of risk in the molecular etiology of breast cancer.
            (Gaikwad et al., 2009a) Download
Endogenous estrogens can be bio-activated to endogenous carcinogens via formation of estrogen quinones. Estrogen-3,4-quinones react with DNA to form mutagenic depurinating estrogen-DNA adducts. The carcinogenicity of endogenous estrogens is related to unbalanced estrogen metabolism leading to excess estrogen quinones and formation of depurinating DNA adducts. The present studies were initiated to confirm that relatively high levels of depurinating estrogen-DNA adducts are present in women at high risk for breast cancer or diagnosed with the disease. These adducts may be biomarkers for early detection of breast cancer risk. The estrogen metabolites, conjugates and depurinating DNA adducts were identified and quantified by using ultraperformance liquid chromatography/tandem mass spectrometry to analyze urine samples from 40 healthy control women, 40 high-risk women and 40 women with newly diagnosed breast cancer. Estrogen metabolism was shifted from protective methoxylation and conjugation pathways in healthy control women towards activating pathways leading to formation of depurinating DNA adducts in women at high risk or with breast cancer. These results support the hypothesis that breast cancer is initiated by mutations derived from depurination of estrogen-DNA adducts. Therefore, relative levels of depurinating estrogen-DNA adducts could become biomarkers for early detection of breast cancer risk and aid in determining preventive strategies.

Formation of diethylstilbestrol-DNA adducts in human breast epithelial cells and inhibition by resveratrol.
            (Hinrichs et al., 2011) Download
Extensive evidence exists that the reaction of estrogen metabolites with DNA produces depurinating adducts that, in turn, induce mutations and cellular transformation. While it is clear that these estrogen metabolites result in a neoplastic phenotype in vitro, further evidence supporting the link between estrogen-DNA adduct formation and its role in neoplasia induction in vivo would strengthen the evidence for a genotoxic mechanism. Diethylstilbestrol (DES), an estrogen analogue known to increase the risk of breast cancer in women exposed in utero, is hypothesized to induce neoplasia through a similar genotoxic mechanism. Cultured MCF-10F human breast epithelial cells were treated with DES at varying concentrations and for various times to determine whether the addition of DES to MCF-10F cells resulted in the formation of depurinating adducts. This is the first demonstration of the formation of DES-DNA adducts in human breast cells. A dose-dependent increase in DES-DNA adducts was observed. Demonstrating that treatment of MCF-10F cells with DES, a known human carcinogen, yields depurinating adducts provides further support for the involvement of these adducts in the induction of breast neoplasia. Previous studies have demonstrated the ability of antioxidants such as resveratrol to prevent the formation of estrogen-DNA adducts, thus preventing a key carcinogenic event. In this study, when MCF-10F cells were treated with a combination of resveratrol and DES, a dose-dependent reduction in the level of DES-DNA adducts was also observed.

Resveratrol prevents estrogen-DNA adduct formation and neoplastic transformation in MCF-10F cells.
            (Lu et al., 2008) Download
Exposure to estrogens is a risk factor for breast cancer. Specific estrogen metabolites may initiate breast cancer and other cancers. Genotoxicity may be caused by cytochrome P450 (CYP)-mediated oxidation of catechol estrogens to quinones that react with DNA to form depurinating estrogen-DNA adducts. CYP1B1 favors quinone formation by catalyzing estrogen 4-hydroxylation, whereas NAD(P)H quinone oxidoreductase 1 (NQO1) catalyzes the protective reduction of quinones to catechols. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) induces CYP1B1 expression through the aryl hydrocarbon receptor (AhR). Resveratrol has anticancer effects in diverse in vitro and in vivo systems and is an AhR antagonist that decreases CYP expression but induces NQO1 expression. The chemopreventive effect of resveratrol on breast cancer initiation was investigated in MCF-10F cells. Its effects on estrogen metabolism and formation of estrogen-DNA adducts were analyzed in culture medium by high-performance liquid chromatography, whereas its effects on CYP1B1 and NQO1 were determined by immunoblotting and immunostaining. The antitransformation effects of resveratrol were also examined. TCDD induced expression of CYP1B1 and its redistribution in the nucleus and cytoplasm. Concomitant treatment with resveratrol dose-dependently suppressed TCDD-induced expression of CYP1B1, mainly in the cytoplasm. Resveratrol dose- and time-dependently induced expression of NQO1. NQO1 is mainly in the perinuclear membrane of control cells, but resveratrol induced NQO1 and its intracellular redistribution, which involves nuclear translocation of nuclear factor erythroid 2-related factor 2. Resveratrol decreased estrogen metabolism and blocked formation of DNA adducts in cells treated with TCDD and/or estradiol. Resveratrol also suppressed TCDD and/or estradiol-induced cell transformation. Thus, resveratrol can prevent breast cancer initiation by blocking multiple sites in the estrogen genotoxicity pathway.

Evaluation of serum estrogen-DNA adducts as potential biomarkers for breast cancer risk.
            (Pruthi et al., 2012) Download
This study was conducted to determine whether the ratio of estrogen-DNA adducts to their respective metabolites and conjugates in serum differed between women with early-onset breast cancer and those with average or high risk of developing breast cancer. Serum samples from women at average risk (n=63) or high risk (n=80) for breast cancer (using Gail model) and women newly diagnosed with early breast cancer (n=79) were analyzed using UPLC-MS/MS. Adduct ratios were statistically compared among the three groups, and the Area Under the Receiver Operating Characteristic Curve (AUC) was used to identify a diagnostic cut-off point. The median adduct ratio in the average-risk group was significantly lower than that of both the high-risk group and the breast cancer group (p values<0.0001), and provided good discrimination between those at average versus high risk of breast cancer (AUC=0.84, 95% CI 0.77-0.90). Sensitivity and specificity were maximized at an adduct ratio of 77. For women in the same age and BMI group, the odds of being at high risk for breast cancer was 8.03 (95% CI 3.46-18.7) times higher for those with a ratio of at least 77 compared to those with a ratio less than 77. The likelihood of being at high risk for breast cancer was significantly increased for those with a high adduct ratio relative to those with a low adduct ratio. These findings suggest that estrogen-DNA adducts deserve further study as potential biomarkers for risk of developing breast cancer.

Estrogen mediation of breast tumor formation involves estrogen receptor-dependent, as well as independent, genotoxic effects.
            (Santen et al., 2009) Download
Long-term exposure to estrogens influences the development of breast cancer in women, but the precise mechanisms involved are not clearly defined. Our working hypothesis is that estrogen modulates this process by two separate processes. One involves the binding of estradiol to estrogen receptor (ER) alpha with stimulation of cell proliferation. Errors in DNA occurring during replication result in fixed mutations when not repaired. The other process results from the formation of genotoxic metabolites of estradiol, which can bind to DNA, cause depurination, and result in mutations. Our collaborative group, funded by a Department of Defense Center of Excellence grant, has examined this hypothesis using a variety of experimental methods. Estradiol and its catechol-estrogen metabolite 4-OH-estradiol causes mutations in cell culture systems and can transform benign MCF-10F cells, allowing them to cause tumors in SCID mice. We have demonstrated loss of heterozygosity and gains and losses of DNA segments by comparative genomic hybridization methodology. The depurinated estradiol-guanine and -adenine adducts are measurable in MCF-7 breast cancer cells in culture and in mouse mammary tissue. The double transgenic, alpha estrogen receptor knockout/Wnt-1 knockin mouse model allows us to dissect out the separate effects of ER-mediated and ER-independent actions of estradiol. Knock out of the ER alpha delays the onset of breast tumors in this model, demonstrating a role of receptor-mediated actions. Oophorectomy delays the onset of tumors and reduces overall incidence, providing evidence for an ER-independent effect. Taken together, these data suggest that both ER-dependent and genotoxic ER-independent effects of estradiol mediate breast cancer development.

Antineoplastic effect of iodine and iodide in dimethylbenz[a]anthracene-induced mammary tumors: association between lactoperoxidase and estrogen-adduct production.
            (Soriano et al., 2011) Download
Several groups, including ours, have reported that iodine exhibited antiproliferative and apoptotic effects in various cancer cells only if this element is supplemented as molecular iodine, or as iodide, to cells that are able to oxidize it with the enzyme thyroperoxidase. In this study, we analyzed the effect of various concentrations of iodine and/or iodide in the dimethylbenz[a]anthracene (DMBA) mammary cancer model in rats. The results show that 0.1% iodine or iodide increases the expression of peroxisome proliferator-activated receptor type γ (PPARγ), triggering caspase-mediated apoptosis pathways in damaged mammary tissue (DMBA-treated mammary gland) as well as in frank mammary tumors, but not in normal mammary gland. DMBA treatment induces the expression of lactoperoxidase, which participates in the antineoplastic effect of iodide and could be involved in the pro-neoplastic effect of estrogens, increasing the formation of DNA adducts. In conclusion, our results show that a supplement of 0.1% molecular iodine/potassium iodide (0.05/0.05%) exert antineoplastic effects, preventing estrogen-induced DNA adducts and inducing apoptosis through PPARγ/caspases in pre-cancer and cancerous cells. Since this iodine concentration does not modify the cytology (histology, apoptosis rate) or physiology (triiodothyronine and thyrotropin) of the thyroid gland, we propose that it be considered as an adjuvant treatment for premenopausal mammary cancer.

Long-term estrogen exposure promotes carcinogen bioactivation, induces persistent changes in gene expression, and enhances the tumorigenicity of MCF-7 human breast cancer cells.
            (Spink et al., 2009) Download
The cumulative exposure to estrogens is an important determinant in the risk of breast cancer, yet the full range of mechanisms involving estrogens in the genesis and progression of breast cancer remains a subject of debate. Interactions of estrogens and environmental toxicants have received attention as putative factors contributing to carcinogenesis. Mechanistic studies have demonstrated interactions between estrogen receptor alpha (ERalpha) and the aryl hydrocarbon receptor (AhR), with consequences on the genes that they regulate. Many studies of ERalpha and AhR-mediated effects and crosstalk between them have focused on the initial molecular events. In this study, we investigated ERalpha- and AhR-mediated effects in long-term estrogen exposed (LTEE) MCF-7 human breast cancer cells, which were obtained by continuous culturing for at least 12 weeks in medium supplemented with 1 nM of 17beta-estradiol (E(2)). With these LTEE cells and with parallel control cells cultured without E(2) supplementation, we performed an extensive study of cytochrome P450 (CYP) induction, carcinogen bioactivation, global gene expression, and tumorigenicity in immunocompromised mice. We found that LTEE cells, in comparison with control cells, had higher levels of AhR mRNA and protein, greater responsiveness for AhR-regulated CYP1A1 and CYP1B1 induction, a 6-fold higher initial level of benzo(a)pyrene-DNA adducts as determined by liquid chromatography tandem mass spectrometry, marked differences in the expression of numerous genes, and a higher rate of E(2)-dependent tumor growth as xenografts. These studies indicate that LTEE causes adaptive responses in MCF-7 cells, which may reflect processes that contribute to the overall carcinogenic effect of E(2).

Lipid peroxidation and DNA adduct formation in lymphocytes of premenopausal women: Role of estrogen metabolites and fatty acid intake.
            (Sun et al., 2012) Download
A diet high in linoleic acid (an ω-6 PUFA) increased the formation of miscoding etheno (ε)--DNA adducts in WBC-DNA of women, but not in men (Nair et al., Cancer Epidemiol Biomark Prev 1997;6:597-601). This gender specificity could result from an interaction between ω-6 PUFA intake and estrogen catabolism, via redox-cycling of 4-hydroxyestradiol (4-OH-E(2) ) and subsequent lipid peroxidation (LPO). In this study, we investigated whether in premenopausal women LPO-derived adducts in WBC-DNA are affected by serum concentration of 2- and 4-hydroxyestradiol metabolites and by fatty acid intake. DNA extracted from buffy coat and plasma samples, both blindly coded from healthy women (N = 124, median age 40 year) participating in the EPIC-Heidelberg cohort study were analyzed. Three LPO-derived exocyclic DNA adducts, εdA, εdC and M(1) dG were quantified by immuno-enriched (32) P-postlabelling and estradiol metabolites by ultra-sensitive GC-mass spectrometry. Mean M(1) dG levels in WBC-DNA were distinctly higher than those of εdA and εdC, and all were positively and significantly interrelated. Serum levels of 4-OH-E(2) , but not of 2-OH-E(2) , metabolites were positively related to etheno DNA adduct formation. Positive correlations existed between M(1) dG levels and linoleic acid intake or the ratios of dietary linoleic acid/oleic acid and PUFA/MUFA. Aerobic incubation of 4-OH-E(2) , arachidonic acid and calf thymus DNA yielded two to threefold higher etheno DNA adduct levels when compared with assays containing 2-OH-E(2) instead. In conclusion, this study is the first to compare M(1) dG and etheno-DNA adducts and serum estradiol metabolites in human samples in the same subjects. Our results support a novel mechanistic link between estradiol catabolism, dietary ω-6 fatty acid intake and LPO-induced DNA damage supported by an in vitro model. Similar studies in human breast epithelial tissue and on amplification of DNA-damage in breast cancer patients are in progress.


 

Inhibition of depurinating estrogen-DNA adduct formation by natural compounds.
            (Zahid et al., 2007) Download
Specific metabolites of estrogens, catechol estrogen-3,4-quinones, if produced in relatively large amounts, can become chemical carcinogens by reacting with DNA to form predominantly depurinating DNA adducts. Estradiol (E2)-3,4-quinone (Q) reacts with DNA to form predominantly the depurinating DNA adducts, 4-hydroxyestradiol (OHE2)-1-N3Ade and 4-OHE 2-1-N7Gua. The depurinating adducts induce mutations by error-prone repair. We have conducted a study in which selected natural chemopreventing agents, N-acetylcysteine (NAcCys), melatonin, reduced lipoic acid, and resveratrol, have been tested for their ability to prevent the reaction of E(2)-3,4-Q with DNA. When DNA was incubated with E(2)-3,4-Q or lactoperoxidase-activated 4-OHE2 in the presence of an antioxidant, the formation of the N3Ade and N7Gua adducts was reduced. E(2)-3,4-Q or lactoperoxidase-oxidized 4-OHE 2 (87 microM final concentration) was incubated with calf-thymus DNA and one of the antioxidants at different ratios (1:0, 1:0.3, 1:1, and 1:3 with respect to E(2)-3,4-Q or 4-OHE2) at 37 degrees C. After 10 h, the DNA was precipitated, and the supernatant was analyzed by using ultraperformance liquid chromatography/tandem mass spectrometry (LC/MS/MS). As anticipated, resveratrol and melatonin did not affect the formation of the depurinating adducts when E(2)-3,4-Q was reacted with DNA in their presence. On the other hand, NAcCys and lipoic acid (reduced form) showed a significant inhibition of the formation of the depurinating adducts by E(2)-3,4-Q. With reaction of lactoperoxidase-activated 4-OHE2 with DNA, resveratrol achieved the highest level of inhibition, NAcCys and reduced lipoic acid produced moderate inhibition, and melatonin had the least inhibition. These results demonstrate that all four selected compounds can inhibit the formation of depurinating estrogen-DNA adducts and set the stage for studies of their ability to inhibit adduct formation and malignant transformation in mammary epithelial cells. This approach is highly useful for identifying agents to prevent the initiation of human cancers, especially breast and prostate cancer.

Prevention of estrogen-DNA adduct formation in MCF-10F cells by resveratrol.
            (Zahid et al., 2008) Download
Resveratrol (Resv), a natural occurring phytolexin present in grapes and other foods, possesses chemopreventive effects revealed by its striking modulation of diverse cellular events associated with tumor initiation, promotion, and progression. Catechol estrogens generated in the metabolism of estrogens are oxidized to catechol quinones that react with DNA to form predominantly depurinating estrogen-DNA adducts. This event can generate the mutations responsible for cancer initiation. In this regard, Resv acts as both an antioxidant and an inducer of the phase II enzyme NAD(P)H:quinone oxidoreductase 1 (NQO1). In this report, we present the effects of Resv on the metabolism of estrogens in normal breast epithelial cells (MCF-10F) treated with 4-hydroxyestradiol (4-OHE(2)) or estradiol-3,4-quinone (E(2)-3,4-Q). Resv induced NQO1 in a dose- and time-dependent manner, but did not affect the expression of catechol-O-methyltransferase. Ultraperformance liquid chromatography/tandem mass spectrometry was used to determine the effects of Resv on estrogen metabolism. Preincubation of the cells with Resv for 48 h decreased the formation of depurinating estrogen-DNA adducts from 4-OHE(2) or E(2)-3,4-Q and increased formation of methoxycatechol estrogens. When Resv was also present with the 4-OHE(2) or E(2)-3,4-Q, even greater increases in methoxycatechol estrogens were observed, and the DNA adducts were undetectable. We conclude that Resv can protect breast cells from carcinogenic estrogen metabolites, suggesting that it could be used in breast cancer prevention.

N-acetylcysteine blocks formation of cancer-initiating estrogen-DNA adducts in cells.
            (Zahid et al., 2010) Download
Catechol estrogens, especially 4-hydroxylated metabolites of 17beta-estradiol (E(2)), are responsible for estrogen-induced carcinogenesis. 4-Hydroxyestradiol (4-OHE(2)), a major metabolite of E(2) formed preferentially by cytochrome P-450 1B1, is oxidized to E(2)-3,4-quinone, which can react with DNA to yield the depurinating adducts 4-OHE(2)-1-N3Ade and 4-OHE(2)-1-N7Gua. The apurinic sites generated by the loss of these depurinating adducts induce mutations that could lead to cancer initiation. In this study, we have evaluated the effects of N-acetylcysteine (NAcCys) on the metabolism of two cell lines, MCF-10F (a normal human breast epithelial cell line) and E6 (a normal mouse mammary epithelial cell line), treated with 4-OHE(2) or its reactive metabolite, E(2)-3,4-quinone. Extensive HPLC with electrochemical detection and UPLC-MS/MS analyses of the cell media demonstrated that the presence of NAcCys very efficiently shifted the estrogen metabolism toward protective methoxylation and conjugation pathways in multiple ways, whereas formation of depurinating DNA adducts was inhibited. Protection by NAcCys seems to be similar in both cell lines, irrespective of their origin (human or mouse) or the presence of estrogen receptor-alpha. This finding suggests that NAcCys, a common dietary supplement, could be used as a potential chemopreventive agent to block the initial step in the genotoxicity caused by catechol estrogen quinones.

Resveratrol and N-acetylcysteine block the cancer-initiating step in MCF-10F cells.
            (Zahid et al., 2011) Download
Substantial evidence suggests that catechol estrogen-3,4-quinones react with DNA to form predominantly the depurinating adducts 4-hydroxyestrone (estradiol)-1-N3Ade [4-OHE(1)(E(2))-1-N3Ade] and 4-OHE(1)(E(2))-1-N7Gua. Apurinic sites resulting from these adducts generate critical mutations that can initiate cancer. The paradigm of cancer initiation is based on an imbalance in estrogen metabolism between activating pathways that lead to estrogen-DNA adducts and deactivating pathways that lead to estrogen metabolites and conjugates. This imbalance can be improved to minimize formation of adducts by using antioxidants, such as resveratrol (Resv) and N-acetylcysteine (NAcCys). To compare the ability of Resv and NAcCys to block formation of estrogen-DNA adducts, we used the human breast epithelial cell line MCF-10F treated with 4-OHE(2). Resv and NAcCys directed the metabolism of 4-OHE(2) toward protective pathways. NAcCys reacted with the quinones and reduced the semiquinones to catechols. This pathway was also carried out by Resv. In addition, Resv induced the protective enzyme quinone reductase, which reduces E(1)(E(2))-3,4-quinones to 4-OHE(1)(E(2)). Resv was more effective at increasing the amount of 4-OCH(3)E(1)(E(2)) than NAcCys. Inhibition of estrogen-DNA adduct formation was similar at lower doses, but at higher doses Resv was about 50% more effective than NAcCys. Their combined effects were additive. Therefore, these two antioxidants provide an excellent combination to protect catechol estrogens from oxidation to catechol quinones.

O-methylation of catechol estrogens by human placental catechol-o-methyltransferase: interindividual differences in sensitivity to heat inactivation and to inhibition by dietary polyphenols.
            (Zhu et al., 2010) Download
The human catechol-O-methyltransferase (COMT) is a polymorphic enzyme that catalyzes the O-methylation of catechol estrogens. Recent animal studies showed that placental COMT is involved in the development of placentas and embryos, probably via the formation of 2-methoxyestradiol. In this study, we analyzed a total of 36 human term placentas to determine their cytosolic COMT activity for the O-methylation of catechol estrogens as well as their sensitivity to inhibition by heat and dietary compounds. Large variations (up to 4-fold) in the COMT activity for the formation of methoxyestrogens were noted with different human placental samples. The cytosolic COMTs in different human placentas also displayed considerable differences in their sensitivity to heat inactivation. This differential sensitivity was not associated with the overall catalytic activity for the O-methylation of catechol estrogen substrates. It was observed that there was a positive correlation (r = 0.760) between the sensitivity of the human placental COMT to heat inactivation and its sensitivity to inhibition by (-)-epigallocatechin-3-gallate (a well known tea polyphenol with COMT-inhibiting activity) but an inverse correlation (r = 0.544) between heat inactivation and inhibition by quercetin (another dietary COMT inhibitor). The differences in inhibition by these two dietary compounds are due to different mechanisms of COMT inhibition involved.

 


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