Original Contribution
Inhibition of catechol-O-methyltransferase increases estrogen–DNA adduct formation

https://doi.org/10.1016/j.freeradbiomed.2007.08.005Get rights and content

Abstract

The association found between breast cancer development and prolonged exposure to estrogens suggests that this hormone is of etiologic importance in the causation of the disease. Studies on estrogen metabolism, formation of DNA adducts, carcinogenicity, cell transformation, and mutagenicity have led to the hypothesis that reaction of certain estrogen metabolites, predominantly catechol estrogen-3,4-quinones, with DNA forms depurinating adducts [4-OHE1(E2)-1-N3Ade and 4-OHE1(E2)-1-N7Gua]. These adducts cause mutations leading to the initiation of breast cancer. Catechol-O-methyltransferase (COMT) is considered an important enzyme that protects cells from the genotoxicity and cytotoxicity of catechol estrogens, by preventing their conversion to quinones. The goal of the present study was to investigate the effect of COMT inhibition on the formation of depurinating estrogen–DNA adducts. Immortalized human breast epithelial MCF-10F cells were treated with 4-OHE2 (0.2 or 0.5 μM) for 24 h at 120, 168, 216, and 264 h postplating or one time at 1–30 μM 4-OHE2 with or without the presence of COMT inhibitor (Ro41-0960). The culture media were collected at each point, extracted by solid-phase extraction, and analyzed by HPLC connected with a multichannel electrochemical detector. The results demonstrate that MCF-10F cells oxidize 4-OHE2 to E1(E2)-3,4-Q, which react with DNA to form the depurinating N3Ade and N7Gua adducts. The COMT inhibitor Ro41-0960 blocked the methoxylation of catechol estrogens, with concomitant 3- to 4-fold increases in the levels of the depurinating adducts. Thus, low activity of COMT leads to higher levels of depurinating estrogen–DNA adducts that can induce mutations and initiate cancer.

Introduction

Prolonged exposure of women to high estrogen levels is associated with an elevated incidence of breast cancer [1], [2], [3], [4], [5]. Experiments on estrogen metabolism [6], [7], [8], [9], [10], formation of DNA adducts [11], [12], [13], [14], [15], [16], [17], mutagenicity [17], [18], [19], [20], [21], cell transformation [22], [23], [24], and carcinogenicity [25], [26], [27], [28] have led to the hypothesis that certain estrogen metabolites, predominantly catechol estrogen-3,4-quinones, react with DNA to cause the mutations leading to the initiation of cancer (Fig. 1) [17]. The reaction of estrone(estradiol)-3,4-quinones [E1(E2)-3,4-Q], derived from 4-OHE1(E2), with DNA produces predominantly depurinating adducts and very small amounts of stable adducts [11], [13], [14], [29].

In extrahepatic tissues, cytochrome P450 (CYP)1A1 and CYP1B1 predominantly metabolize the natural estrogens E1 and E2 to 2- and 4-catechol estrogens (CE), respectively [30], [31], [32], which can be competitively oxidized to their respective semiquinones and quinones. In general, the CE are inactivated by conjugating reactions, such as glucuronidation and sulfation. A common pathway of inactivation in extrahepatic tissues, however, occurs by O-methylation catalyzed by the ubiquitous catechol-O-methyltransferase (COMT) [33]. If the formation of E1 and E2 is excessive, due to overexpression of aromatase and/or the presence of excess sulfatase that converts stored E1 sulfate to E1, increased formation of CE is expected (Fig. 1). In particular, the presence and/or induction of CYP1B1 and other 4-hydroxylases could dramatically increase the formation of 4-OHE1(E2). Thus, conjugation of 4-OHE1(E2) via methylation can become insufficient, and competitive oxidation of 4-OHE1(E2) to E1(E2)-3,4-Q could be more abundant [34]. The increased level of quinones would generate more reaction with DNA at the N-3 of adenine (Ade) and N-7 of guanine (Gua) to form depurinating adducts (Fig. 1) [17], [29], [34]. These adducts are lost from DNA by destabilization of the glycosyl bond. The apurinic sites generated in the DNA can produce mutations by error-prone repair [18], [19], [20], which in turn can lead to initiation of cancer.

The phase II enzyme COMT is considered to be a key enzyme in decreasing the effects of 4-OHE1(E2) by converting the catechol estrogens into the corresponding methoxy derivatives [33]. COMT is an intracellular enzyme that is present as both soluble and membrane-bound forms encoded by the same gene with different transcription start sites [35]; the soluble form is the major one in most organs [33]. COMT activity can be altered by either endogenous factors, such as genetic polymorphisms and levels of expression, or exogenous factors such as inhibition by environmental compounds. Genetic epidemiology studies have proposed a possible correlation between the low activity allele (COMTLL) and increased breast cancer risk [36], [37], [38].

COMT activity can be inhibited by many natural and synthetic compounds [33], [39], [40]. Ro41-0960 is a nitrocatechol-type inhibitor of COMT that inhibits methylation of catechol estrogens. It is a poor substrate for COMT, but binds tightly to catalytic sites of the enzyme, thus inhibiting methylation of other substrates without depleting cofactors [41], [42]. We hypothesize that COMT inhibition decreases inactivation of CE, which may in turn lead to increased formation of CE-Q and DNA damage that initiates cancer.

To fully understand how estrogens can become carcinogenic in the human breast through metabolic activation to CE-Q an experimental system is required in which estrogens or their metabolites (e.g., 4-OHE2) would induce transformation phenotypes in human breast epithelial cells in vitro that are indicative of neoplasia. Data from a recent study showed that successive treatment of MCF-10F cells with 4-OHE2 induced mutations, cell transformation, and cancer [21], [24]. The ERα-negative MCF-10F cell line is a good experimental model for researching the carcinogenicity and mutagenic potential of 4-OHE2. To investigate the implications of possible COMT inhibition by Ro41-0960 and increased formation of depurinating adducts, the cells were preincubated with 3 μM Ro41-0960 and then treated with 4-OHE2 (0.2–30 μM) for 24 h. The profile of 4-OHE2 metabolites, conjugates, and depurinating DNA adducts was determined in cell culture medium by HPLC equipped with a multichannel electrochemical detector (ECD) and validated by ultraperformance liquid chromatography (UPLC)-MS/MS techniques. This is the first report on the metabolic profile of 4-OHE2 in MCF-10F cells treated in a dose–response manner.

Section snippets

Chemicals and reagents

4-OHE2 and all standards were synthesized in our laboratory, as previously described [13], [43], [44], [45]. Ro41-0960 and all other chemicals were purchased from Sigma (St. Louis, MO). MCF-10F cells were obtained from the ATCC (Rockville, MD).

Cell culture and treatment

MCF-10F cells were cultured in phenol red DMEM/F12 (1/1) medium containing 20 ng/ml epidermal growth factor, 0.01 mg/ml insulin, 500 ng/ml hydrocortisone, 5% horse serum, and 100 μg/ml penicillin/streptomycin mixture and maintained in a humidified

Results and discussion

To examine the profile of estrogen metabolism in MCF-10F cells, an HPLC method with a CoulArray ECD [10] was used to quantify the relative concentrations of estrogen metabolites, conjugates, and depurinating DNA adducts. Standard solutions of each compound were combined to generate equimolar mixtures containing varying concentrations of each estrogen standard and injected onto the column. These standard solutions were then used to generate calibration curves. Standard curves were linear between

Acknowledgments

This research was supported by U.S. Public Health Service Grant P01 CA49210 from the National Cancer Institute and the U.S. Army Breast Cancer Research Program Grant DAMD 17-03-1-0229. Core support at the Eppley Institute was provided by Grant P30 CA36727 from the National Cancer Institute.

References (51)

  • Y.S. Ding et al.

    Mapping catechol-O-methytransferase in vivo: initial studies with [18F]Ro41-0960.

    Life Sci.

    (1995)
  • M. Saeed et al.

    Synthesis of the catechols of natural and synthetic estrogens by using 2′-iodoxybenzoic acid (IBX) as the oxidizing agent

    Steroids

    (2005)
  • J.F. Dorgan et al.

    Relation of prediagnostic serum estrogen and androgen levels to breast cancer risk

    Cancer Epidemiol. Biomarkers Prev.

    (1996)
  • H.V. Thomas et al.

    A prospective study of endogenous serum hormone concentrations and breast cancer risk in postmenopausal women on the island of Guernsey

    Br. J. Cancer

    (1997)
  • S.E. Hankinson et al.

    Plasma sex steroid hormone levels and risk of breast cancer in postmenopausal women

    J. Natl. Cancer Inst.

    (1998)
  • M. Kabuto et al.

    A prospective study of estradiol and breast cancer in Japanese women

    Cancer Epidemiol. Biomarkers Prev.

    (2000)
  • Endogenous sex hormones and breast cancer in postmenopausal women: reanalysis of nine prospective studies

    J. Natl. Cancer Inst.

    (2002)
  • B.T. Zhu et al.

    Functional role of estrogen metabolism in target cells: review and perspectives

    Carcinogenesis

    (1998)
  • E.L. Cavalieri et al.

    Imbalance of estrogen homeostasis in kidney and liver of hamsters treated with estradiol: implications for estrogen-induced initiation of renal tumors

    Chem. Res. Toxicol.

    (2001)
  • P. Devanesan et al.

    Catechol estrogen metabolites and conjugates in mammary tumors with hyperplastic tissue from estrogen receptor-α knock-out (ERKO)/Wnt-1 mice: implications for initiation of mammary tumors

    Carcinogenesis

    (2001)
  • E.L. Cavalieri et al.

    Catechol estrogen metabolites and conjugates in different regions of the prostate of Noble rats treated with 4-hydroxyestradiol: implications for estrogen-induced initiation of prostate cancer

    Carcinogenesis

    (2002)
  • E.G. Rogan et al.

    Relative imbalances in estrogen metabolism and conjugation in breast tissue of women with carcinoma: potential biomarkers of susceptibility to cancer

    Carcinogenesis

    (2003)
  • E.L. Cavalieri et al.

    Molecular origin of cancer: catechol estrogen-3,4-quinones as endogenous tumor initiators

    Proc. Natl. Acad. Sci. USA

    (1997)
  • Y. Markushin et al.

    Spectral characterization of catechol estrogen quinone (CEQ)-derived DNA adducts and their identification in human breast tissue extract

    Chem. Res. Toxicol.

    (2003)
  • K.-M. Li et al.

    Metabolism and DNA binding studies of 4-hydroxyestradiol and estradiol-3,4-quinone in vitro and in female ACI rat mammary gland in vivo

    Carcinogenesis

    (2004)
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