Article pubs.acs.org/crt
Oxidative DNA Damage Following Microsome/Cu(II)-Mediated Activation of the Estrogens, 17β-Estradiol, Equilenin, and Equilin: Role of Reactive Oxygen Species† Wendy A. Spencer,‡ Manicka V. Vadhanam,‡ Jeyaprakash Jeyabalan,‡ and Ramesh C. Gupta*,‡,§ ‡
James Graham Brown Cancer Center, and §Department of Pharmacology and Toxicology, University of Louisville, Louisville, Kentucky 40202, United States ABSTRACT: Experimental and epidemiological data associate the exposure of estrogens to cancer development in several tissues, particularly, the breast, endometrium, liver, and kidney. One plausible mechanism of estrogen-mediated carcinogenicity is DNA damage by redox cycling of estrogen catechols. Reports have shown that metabolism of estrogens results in 2- and 4-hydroxylation to catechol metabolites which can then redox cycle. We examined the capacity of the endogenous estrogen, 17β-estradiol, and two equine estrogens which formulate a significant proportion of hormone replacement drugs, equilenin and equilin, to induce oxidatively generated DNA damage. Microsome/Cu(II)-mediated activation of all three estrogens resulted in numerous oxidation DNA adducts, as detected by 32Ppostlabeling/TLC. Essentially the same DNA oxidation pattern was also found when catechol estrogens were incubated with DNA in the presence of Cu(II) suggesting that redox cycling of catechol estrogens mediates the formation of these DNA adducts. Since the oxidation patterns induced by estrogen catechols and other chemically diverse catechols were chromatographically identical to those generated by Fenton-type chemistry and these adducts were inhibited by known ROS modifiers (up to 96%), this oxidatively generated DNA damage is believed to be the product of the attack of free radicals on DNA, rather than direct addition of the estrogen quinones. These data support a mechanistic role by endogenous and synthetic estrogens to induce oxidative DNA damage in addition to specific DNA adducts.
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INTRODUCTION Substantial epidemiological as well as experimental evidence exists indicating a causal role of estrogens in cancer development in estrogen-responsive tissues, especially the breast and endometrium. Estrogens have long been known to cause a variety of cancer types in laboratory animals, including rat mammary,1,2 hamster kidney,3,4 mouse uterus,5 and rat pituitary.6 Moreover, risk factors associated with breast and endometrial cancers in humans, such as the early onset of menarche, age of first pregnancy, and late onset of menopause, are strongly linked with extended exposure to estrogens.7 Reduction of estrogen levels by removal of the ovaries prior to menopause has been found to significantly reduce breast cancer risk.8 Further estrogen levels have been found to be several times higher in both breast and endometrial cancer tissues when compared to adjacent normal tissues of the same subjects or blood.9 Exogenous exposure to estrogens, as oral contraceptives or estrogen-replacement therapy (ERT) following menopause, has also been linked to increased risk of cancer of both breast, up to 2-fold, and endometrial cancers, as much as 10-fold.10−14 Notably, the benefits of ERT and HRT have been further challenged as two arms of the Women’s Health Initiative Study involving estrogen and progestin treatment in © 2011 American Chemical Society
postmenopausal women were halted prematurely due to significant increases in coronary heart disease, breast cancer, stroke, pulmonary embolism, and vascular dementia.15 In fact, the National Toxicology Program of the NIEHS recognizes steroidal estrogens, including endogenous sources and those contained in HRTs, as human carcinogens and causative agents for breast and endometrial cancers.15 Although considerable efforts have been made to understand the role of estrogens in cancer development, their mechanisms remain unclear. One potential mechanism of estrogen-mediated carcinogenesis originates from its hormonal effects via receptormediated pathways.15,16 Estrogens, by this mechanism, are believed to cause over stimulation of the pituitary gland resulting in excessive production of gonadotrophins and thus over stimulation of the target organs and neoplasia. However, this is not believed to be the only mechanism since the hormonal potencies of estrogens do not correlate with tumor incidence.14 A second plausible mechanism of estrogenmediated cancer development is redox cycling of estrogen metabolites resulting in the production of DNA-damaging Received: August 18, 2011 Published: November 29, 2011 305
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Figure 1. Structures of endogenous and equine estrogens found in estrogen replacement therapy (ERT) formulations. Relatively high concentrations of copper and estrogens, compared to the concentrations present in biological systems, were used in these studies in order to generate readily detectable and measurable levels of these oxidatively generated DNA adducts, as well as reliably measure the effect of ROS modifiers. Nonenzymatic DNA Damage. St-DNA (300 μg/mL) was incubated (37 °C, 1 h) with E2, 2E2 (2-hydroxy-estradiol), 4E2 (4hydroxy-estradiol), 2E1 (2-hydroxy-estrone), or 4E1 (4-hydroxyestrone) (100 μM) in the presence or absence of CuCl2 (100 μM) in 10 mM Tris·HCl, pH 7.4. The reaction was terminated by extraction of the reaction mixture with chloroform/isoamylalcohol (24:1), followed by ethanol precipitation of DNA. In order to identify and determine the possible role of various ROS in the generation of these DNA adducts during Cu(II)-mediated redox cycling of catechol estrogens, intervention with known ROS scavengers/modifiers was carried out as described above using 4E2 (100 μM) as the substrate in the presence of CuCl2 (100 μM) and either 100 mM sodium azide, 10 mM tiron, 200 U/mL catalase, or 150 μM bathocuproine. All ROS modifiers were dissolved in HPLC-grade water except bathocuproine, which was dissolved in dimethyl sulfoxide. 32 P-Postlabeling Analysis. Unidentified Oxidative DNA Adducts. Analysis of the unidentified oxidative DNA adducts was as described elsewhere.24 Briefly, DNA (6 μg) was hydrolyzed to 3′monophosphates with a mixture of micrococcal nuclease and spleen phosphodiesterase, and adducts were enriched by treatment with nuclease P1. Adducts were 5′-32P-labeled in the presence of molar excess of [γ-32P]ATP (1200 adducts/106 nucleotides), while Eqn and Eq resulted in 2-fold and 7-fold lower levels of DNA damage, respectively, at this concentration. Species-Dependent Metabolism of Estrogens. In order to determine if there were species differences in estrogen metabolism, E2, Eqn, and Eq (50 μM) were reacted with DNA in the presence of liver microsomes from Sprague−Dawley rats, CD-1 mice, or humans (200 μg/mL) and an NADPHregenerating system, followed by the addition of CuCl2. Following 32P-postlabeling of the DNA, an essentially identical oxidatively generated DNA oxidation pattern, as found using rat liver microsomes, was observed by all three estrogens with either mouse or human microsomes; 8-oxodG was not analyzed in this study. However, metabolism with rat liver microsomes resulted in the highest levels of oxidative DNA damage for all three estrogens tested, yielding adduct levels of 881 ± 60, 464 ± 20, and 106 ± 8 adducts/106 nucleotides for E2, Eqn, and Eq, respectively (Figure 7). Mouse and human microsomes resulted in 1.5 to 3.5- and 5 to 20-fold lower levels of oxidative DNA damage, respectively. Gender-Dependent Metabolism of E2. To determine if there were gender differences in estrogen-induced oxidative DNA damage, E2 (50 μM) was metabolized, in the presence of an NADPH-regenerating system and CuCl2, with liver microsomes (200 μg/mL) prepared from both male and female Sprague−Dawley rats. No differences in the DNA decomposition pattern were observed (data not shown). Although male microsome activation of E2 resulted in slightly higher oxidation adduct levels (30%), the difference in levels was statistically insignificant (p > 0.05).
4E2 oxidative DNA damage, several ROS modifiers, sodium azide (a singlet oxygen and hydroxyl radical scavenger), tiron (a superoxide scavenger), catalase (an enzyme that catalyzes the decomposition of hydrogen peroxide to water and oxygen), and bathocuproine (a Cu(I)-specific chelator, used in previous studies at similar concentrations),27,28 were added to the standard reaction mixture. Although no qualitative differences were observed in the DNA oxidation pattern following intervention with any of the modifiers tested, significant quantitative differences in 8-oxo-dG and the unidentified oxidative DNA adduct levels were observed (Figure 5). All
Figure 5. Effect of ROS modifiers on Cu(II) (100 μM)-mediated oxidatively generated DNA damage by 4E2 (100 μM). Incubation of DNA with 4E2 and the indicated ROS modifiers was carried out at 37 °C, pH 7.4, for 1 h. Levels of the oxidation DNA adducts were determined as the means of four to six replicates ± SE. * Indicates significantly different from vehicle at P ≤ 0.05. Specific conditions are described in the text. The modifiers and their target ROS were bathocuproine, Cu(I); catalase, hydrogen peroxide; tiron, superoxide; and sodium azide, singlet oxygen and hydroxyl radical. Mean levels of unidentified oxidation DNA adducts and 8-oxodG in 4E2 treated DNA were 292 ± 28 and 146 ± 13 adducts/106 nucleotides, respectively.
four ROS modifiers tested significantly inhibited (36−96%) oxidative DNA damage induced by Cu(II) catalysis of 4E2. Optimization of Microsome/Cu(II) Model Conditions. Different microsome concentrations (0.1, 0.3, 0.5, and 1 mg/ mL) and incubation times (0.5, 1, 2, and 3 h) were tested to optimize the conditions for estrogen metabolism using the microsome/Cu(II)-mediated two-step system. Microsomal protein concentrations ranging from 0.1 to 0.5 mg/mL did not result in any significant difference in oxidative DNA adduct levels (data not shown). However, higher protein concentrations (≥1 mg/mL) resulted in significantly (50%) diminished adduct levels presumably due to protein/DNA competition for the reactive species. On the basis of these data, a protein concentration of 200 μg/mL was selected for the remainder of the studies. Levels of oxidative DNA adducts by E2-mediation peaked (973 ± 80 adducts/106 nucleotides) (8oxodG was not analyzed in this experiment), following a 1 h microsomal incubation and steadily declined thereafter yielding levels of 712 ± 57 and 569 ± 89 adducts/106 nucleotides following incubation times of 2 and 3 h, respectively. The decline in adduct levels following longer incubation times may be due to loss or instability of the DNA-reactive species over
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DISCUSSION We have detected numerous unidentified oxidatively generated DNA adducts following microsomal/Cu2+-mediated activation of the endogenous estrogen, E2, and the equine estrogens Eqn and Eq commonly used in ERT. These adducts were chromatographically identical to those found following Cu(II)-mediated activation of the catechol estrogen metabolites with the exception of an additional adduct spot observed following Cu(II) catalysis of the catechol estrogens. Moreover, an identical DNA oxidation pattern was found following transition metal activation of numerous structurally diverse catechols, including PCB diols,29 PAH diols,30 and catecholamine neurotransmitters such as dopamine and epinephrine27 as well as the Fenton-type reaction (Cu(II)/H2O2).31 In fact, a common mechanism of catechol oxidation for natural and synthetic estrogens, benzene, dopamine, and naphthalene has previously been proposed to proceed through a semiquinone/ quinone mechanism, and analogous DNA adducts formed by the quinones of these compounds have been observed for all of 309
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Figure 6. Effect of estrogen concentration on oxidative DNA damage following the incubation of DNA (300 μg/mL) with 17β-estradiol (E2), equilenin (Eqn), or equilin (Eq) (2, 10, and 50 μM) in the presence of rat liver microsomes (200 μg/mL), an NADPH-regenerating system, and CuCl2 (100 μM). Incubation of DNA with the indicated estrogens, rat liver microsomes, and an NADPH-regenerating system was carried out at 37 °C, pH 7.4, for 1 h followed by further incubation for 4 h following the addition of CuCl2. The reaction was terminated by adding 20 mM EDTA. DNA adduct levels were determined as the average adduct level of 3 to 4 replicates minus the average adduct level of vehicle treated samples. The standard error ranged from 3 to 17% for unidentified oxidative adducts and 1 to 20% for 8-oxodG. Mean levels of unidentified oxidation DNA adducts and 8-oxodG in vehicle-treated DNA were 7 ± 1 and 53 ± 2 adducts/106 nucleotides, respectively.
these compounds.17 The subject of these quinone covalent stable estrogen DNA adducts and depurinating lesions has been previously addressed in a recent review by Dr. Cavalieri and coworkers.17 Taken together, these findings strongly support the role of common redox cycling intermediates in the formation of these oxidative DNA adducts. Although most of these oxidation DNA adducts have not yet been structurally identified, their characterization as oxidatively generated DNA lesions is based on several compelling observations: (1) ability to migrate under low-salt conditions while adducts resulting from direct interaction of catechols (e.g., chlorinated benzoquinones), being lipophilic, required high ionic-strength solvents containing urea;32 (2) a chromatographically identical oxidation pattern as that produced from typical Fenton-type chemistry during the reduction of hydrogen peroxide in the presence of reduced transition metals; (3) proximity to known oxidative markers including 5-hydroxy-2′-deoxyuridine, thymidine glycol, and 8-oxodG following TLC;24 (4) ability to be inhibited in the presence of ROS scavengers/mediators, including sodium azide, catalase, and tiron;27 and (5) chromatographically similar DNA oxidation patterns have also been found with numerous structurally diverse redox-active agents.29,30 Unpublished studies from this laboratory have further indicated that the majority of these DNA adducts are derivatives of both mono-
and dinucleotides (Ravikumar, M. N. V., and Gupta, R. C., unpublished data). The proposed mechanism of this oxidative DNA damage is believed to occur via redox cycling of estrogen catechols and quinones via semiquinone intermediates (Figure 8). We have found that metabolism of the estrogens to catechol intermediates, through the action of cytochrome P450 as the first step, and treatment with the transition metal Cu(II), as the second step, are required for induction of the oxidative DNA damage. Further, studies here examining the effects of the ROS modifiers, sodium azide, catalase, tiron, and bathocuproine on Cu(II)-catalyzed 4E2 oxidation and subsequent oxidative DNA damage implicate a role of singlet oxygen and the hydroxyl radical, hydrogen peroxide, superoxide, and Cu(I), respectively, in estrogen catechol-mediated oxidative DNA damage. These observations are comparable to those found in our previously published studies of other redox-cycling catecholic species.27,28 Several lines of evidence have been identified for the potential role of oxidative DNA damage and the formation of oxidatively derived stable DNA adducts resulting from redox cycling of catechol estrogens such as 4E2 in estrogen-mediated carcinogenesis. The formation of 4E2 has been correlated with an organ’s increased susceptibility to estrogen-induced carcinogenesis in rat models, and importantly human breast tumor tissues showed elevated levels of 4-hydroxylase activity when 310
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Figure 7. Effect of species differences on oxidative DNA damage following the incubation of DNA (300 μg/mL) with 17β-estradiol (E2), equilenin (Eqn), or equilin (Eq) (50 μM) in the presence of Sprague−Dawley rat, CD-1 mouse, or human liver microsomes (200 μg/mL), an NADPHregenerating system, and CuCl2 (100 μM). Incubation of DNA with the indicated estrogens, liver microsomes, and an NADPH-regenerating system was carried out at 37 °C, pH 7.4, for 1 h followed by further incubation for 4 h following the addition of CuCl2. The reaction was terminated by adding 20 mM EDTA. DNA product levels were determined as the average levels of 3 to 4 replicates ± SE. 8-OxodG levels are not included.
compared to that of normal human breast tissue.33 Further, studies from our own laboratory using a rodent model have shown that a tumorigenic dose of E234 substantially increases the levels of stable oxidative DNA adducts such as 8-oxodG as well as other oxidatively derived DNA adducts.35 These data support the plausible role of the generation of these stable oxidative DNA adducts in estrogen-mediated carcinogenesis. The specific catechols involved in the reaction are believed to be different depending on the estrogen used. Studies have shown that cytochrome P450-mediated aromatic hydroxylation of E2 to the catechol metabolites, 2E2 and 4E2, is the major metabolic pathway for endogenous estrogens.36,37 Metabolism of Eq, an equine estrogen present in ERT formulations, also results in the formation of 2- and 4-catechol metabolites, while Eqn, also present in ERT formulations, is hydroxlyated exclusively at the C-4 position.21,38,35 Our studies with Cu(II)-mediated activation of the catechol estrogens 2E2, 4E2, 2E1, and 4E1 indicate a modestly lower (10−45%) redox activity for 4- versus 2-hydroxylated catechols and for 2- and 4E1 versus 2- and 4E2 (Table 1). This finding is supported by our enzymatic studies in that E2 metabolism produced greater levels (up to 10-fold) of oxidative damage than either Eqn or Eq as 2-hydroxlyation predominates over 4hydroxlylation for E2 but not for Eq or Eqn in rats.22 Our studies also showed that Eqn induced up to 4-fold higher oxidative DNA damage than Eq. This difference in oxidative DNA damage between Eqn and Eq is most likely due to metabolic rate differences and is supported by a study in which 4-hydroxyequilin-o-quinone was 3- to 9-fold less effective at
consuming NAD(P)H than 4-Eqn-o-quinone.22 Further, 4-Eqno-quinone (t1/2 = 2.3 h) has been found to be more stable than either 2-Eq-o-quinone or 4-Eq-o-quinone, which itself readily isomerizes to 4-Eqn-o-quinone, thus indicating a potential for increased redox activity of Eqn over Eq.15 Species differences in estrogen metabolism have been reported;36 however, the mechanisms are complex. In this regard, we have detected significant differences in the level of oxidative DNA damage but not the oxidation pattern induced by microsome activation of estrogens. Rat liver microsome activation of all three estrogens, E2, Eqn, and Eq, resulted in 1.5 to 3.5-fold greater oxidative DNA damage than mouse liver microsomes and 5 to 20-fold greater adduct levels than human liver microsomes. The relative potencies of the estrogens to mediate oxidative DNA damage was the same for all species tested, i.e., E2 > Eqn > Eq. Numerous isoforms of cytochrome P450 in rodents and humans have been identified in several tissue types, in addition to the liver, which can metabolize estrogens to catechol metabolites.39 In humans, CYP1A1 and 1B1 are believed to be the primary extra-hepatic isozymes responsible for estrogen metabolism, while CYP1A2 and 3A4 are the major isozymes involved in the liver metabolism of estrogens.36,37 In general, 2-hydroxylation of E2 predominates in the liver, while 4-hydroxylation predominates in extra-hepatic tissues such as the breast and uterus. This difference may play an important role in estrogen-mediated carcinogenesis as these are the estrogen-sensitive tissues in humans. Although oxidative DNA product levels were greatly dependent on species, they appeared to be independent of gender. 311
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Figure 8. Proposed mechanism of estrogen-mediated oxidative DNA damage.
Oxidation of estrogen catechols to o-quinones occurs both enzymatically, via oxidases and peroxidases, and nonenzymatically, in the presence of transition metals such as copper.18 Studies have indicated that the endogenous catechol estrogens, such as 4E2 and 2E2, require either enzymatic or metal catalysis for the formation of oxidative DNA bases and strand breaks,40,41 while the Eqn catechol, 4Eqn, autoxidizes to an oquinone inducing oxidative DNA damage in the absence of these catalysts.19,22 4Eqn has also been found to induce DNA strand breaks as well as oxidative DNA damage in the form of 8-oxodG and 8-oxodA.19 Treatment of DNA with 4Eqn alone resulted in increased DNA strand breaks and 8-oxodG levels above that of the vehicle, while the addition of Cu(II) and NADH further enhanced DNA strand breaks but only slightly increased 8-oxodG levels. In our studies, microsome-mediated activation of E2, Eqn, or Eq resulted in no significant increase in oxidative DNA damage in the absence of Cu(II) over that of the vehicle. Further, no significant increase of oxidative DNA adducts above that of the vehicle was found following the incubation of DNA with 4E2 alone. Thus, we conclude that a catalyst, in this case, copper, is required for the induction of the
oxidative DNA adducts from catechols of E2. We hypothesize that copper mediates estrogen-induced oxidative damage by reacting with hydrogen peroxide, produced from dismutation of superoxide, a byproduct of redox cycling of the estrogen quinones and semiquinones, resulting in the production of hydroxyl radicals, singlet oxygen, and metal-peroxide complexes previously shown to damage DNA.42 Copper is an essential element in the human diet and is required for normal enzymatic function. Imbalance of this trace mineral has been associated with the pathogenesis of numerous diseases including cancer.43 The total body burden of copper has been estimated to be between 80 and 120 mg in a healthy adult with approximately 20% of this amount stored in the nucleus.43,44 Studies have also shown that Cu(II) has a high affinity to DNA, preferentially binding to guanine residues at the N7 position.43 This Cu(II)−DNA interaction has been shown to promote DNA oxidation and the resulting damage enhanced by packaging of DNA as a nucleosome.43,45 Thus, the availability and reactivity of copper coupled with its close proximity to DNA makes this transition metal a likely and pertinent participant in ROS-mediated DNA damage. 312
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ABBREVIATIONS ERT, estrogen replacement therapy; E2, 17β-estradiol; E1, estrone; ROS, reactive oxygen species; Eqn, equilenin; Eq, equilin; PEI, polyethyleneimine; RAL, relative adduct level; 2E2, 2-hydroxy-estradiol; 4E2, 4-hydroxy-estradiol; 2E1, 2-hydroxyestrone; 4E1, 4-hydroxy-estrone
Recent in vivo studies with catechol estrogens also support the role of redox cycling of quinone and semiquinone intermediates and DNA damage as a mechanism of estrogenmediated carcinogenesis. Intramammillary injection of 4Eqn into Sprague−Dawley rats resulted in both DNA strand breaks and oxidized bases and increased the formation of in 8-oxodG and 8-oxodA in mammary tissues of exposed rats.21 In recent studies from our laboratory, numerous polar, presumably oxidative, DNA adducts, chromatographically similar but not identical to those found in vitro, have been detected in rat mammary and liver tissues following i.p. injection with E2 and 4E2 (Arif, J. M., Vadhanam, M. V., and Gupta, R. C., unpublished data). Additional studies support the role of 4hydroxylated but not 2-hydroxylated metabolites in estrogenmediated carcinogenesis since 4E2 has been shown to predominate in all tissues known to be susceptible to E2induced tumorigenesis, including hamster kidney, mouse uterus, and rat pituitary as opposed to resistant tissues such as the liver.14,46 Further, 4E2 has been shown to induce a greater incidence of uterine tumors (66%) in mice as compared to 2E2 (12%) or E2 (7%).5 In a hamster kidney tumor model, 4E2 was also found to be carcinogenic, while 2E2 was not.47 This lack of carcinogenicity of 2E2 in this model contributed to its rapid methylation and metabolic clearance as opposed to its intrinsic carcinogenic activity. Recent evidence suggests that the estrogen receptor can potentiate the role of catechol estrogens, specifically the 4hydroxlylated metabolites, in oxidative DNA damage. Both, 4E2 and 4Eqn were found to induce DNA single-strand cleavage in estrogen receptor-positive and -negative cells. However, the damage was significantly higher in cells containing estrogen receptors.15,20,48 It is hypothesized that catechol estrogens bind to estrogen receptors and are carried directly to estrogensensitive genes where they can redox cycle resulting in selective DNA damage. In conclusion, we have shown that metabolism of endogenous estrogens as well as those commonly found in ERT formulations can induce oxidative DNA damage as detected by the formation of several oxidation DNA adducts, including 8-oxodG. This mechanism of oxidative DNA damage not only requires metabolism of the estrogens to catechol intermediates but also requires catalysis by transition metals and is mediated by the ROS, singlet oxygen, hydroxyl radical, superoxide, and Cu(I).
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AUTHOR INFORMATION
Corresponding Author
*304E, Delia Baxter Research Building, 580 S. Preston St., Louisville, KY 40202, USA. Phone: 502-852-3682. Fax: 502852-3662. E-mail:
[email protected]. Funding
This work was supported by the USPHS grants CA-118114, CA-125152, CA-90892, and CA-92758, Kentucky Lung Cancer Research Program Cycle 7, and by Agnes Brown Duggan Endowment Funds. R.C.G. holds the Agnes Brown Duggan Chair in Oncological Research. Notes †
Part of this work was conducted at the Department of Preventive Medicine and Environmental Health, and Graduate Center for Toxicology, University of Kentucky Medical Center, 354 Health Sciences Research Building, Lexington, KY 40536. 313
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Chemical Research in Toxicology
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dx.doi.org/10.1021/tx200356v | Chem. Res. Toxicol. 2012, 25, 305−314
