Original ContributionInhibition of catechol-O-methyltransferase increases estrogen–DNA adduct formation
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.
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