4-hydroxyestradiol and estradiol-3-4-quinone

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.

Topics: Acetylcysteine; Animals; Antioxidants; Breast; Breast Neoplasms; Cell Line; DNA Adducts; Estradiol; Estrogens, Catechol; Female; Humans; Mammary Glands, Animal; Mammary Neoplasms, Experimental; Mice

Metabolic conversion of endogenous estrogens, estradiol (E2) and estrone (E1), to the catechol estrogens 4-hydroxyE1(E2) [4-OHE1(E2)] has been implicated in the initiation of cancer in rodents and humans. Evidence collected in our laboratories has shown that 4-OHE1(E2) are enzymatically oxidized to E1(E2)-3,4-quinones [E1(E2)-3,4-Q], which have the potential to damage DNA by forming predominantly depurinating adducts, 4-OHE1(E2)-1-N3Ade and 4-OHE1(E2)-1-N7Gua, leading to the accumulation of mutations and probably cell transformation. The human breast epithelial cell line MCF-10F has been transformed by treatment with E2 or 4-OHE2. We have used MCF-10F cells to study the presence of adducts and conjugates after treatment with 4-OHE2. To mimic the intermittent exposure of breast cells to endogenous estrogens, MCF-10F cells were treated with 1 microM 4-OHE2 for a 24-h period at 72, 120, 192 and 240 h postplating. Culture media were collected at each point, extracted by solid-phase extraction and analyzed by HPLC connected with a multichannel electrochemical detector and/or ultraperformance liquid chromatography/tandem mass spectrometry. Media from successive treatments with 4-OHE2 showed the formation of methoxy and cysteine conjugates, and the depurinating adducts 4-OHE1(E2)-1-N3Ade. The amount of 4-OHE1(E2)-1-N3Ade adducts was higher after the third treatment; smaller amounts of the 4-OHE1(E2)-1-N7Gua adducts were detected after the second and third treatments. These 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. This DNA damage can play an important role in the 4-OHE2-induced mutations and transformation of MCF-10F cells to malignant cells.

Topics: Adenine; Breast; Cells, Cultured; Chromatography, High Pressure Liquid; DNA; DNA Adducts; DNA Damage; Estradiol; Estrogens, Catechol; Guanine; Humans; Molecular Structure; Solid Phase Extraction; Tandem Mass Spectrometry

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.

Topics: Animals; Antioxidants; Biological Products; Cattle; DNA; DNA Adducts; Estradiol; Estrogens, Catechol; Molecular Structure

Strong evidence supports the idea that specific metabolites of estrogens, mainly catechol estrogen-3,4-quinones, can react with DNA to become endogenous initiators of breast, prostate, and other human cancers. Oxidation of the catechol estrogen metabolites 4-hydroxyestradiol (4-OHE2) and 2-OHE2 leads to the quinones, estradiol-3,4-quinone (E2-3,4-Q) and estradiol-2,3-quinone (E2-2,3-Q), respectively. The reaction of E2-3,4-Q with DNA affords predominantly the depurinating adducts 4-OHE2-1-N3Ade and 4-OHE2-1-N7Gua, whereas the reaction of E2-2,3-Q with DNA yields the newly synthesized depurinating adduct 2-OHE2-6-N3Ade. The N3Ade adducts are lost from DNA by rapid depurination, while the N7Gua adduct is lost from DNA with a half-life of approximately 3 h at 37 degrees C. To compare the relative reactivity of E2-3,4-Q and E2-2,3-Q, the compounds were reacted individually with DNA for 0.5-20 h at 37 degrees C, as well as in mixtures (3:1, 1:1, 1:3, and 5:95) for 10 h at 37 degrees C. Depurinating and stable adducts were analyzed. In similar experiments, the relative reactivity of 4-OHE2 and 2-OHE2 with DNA was determined after activation by lactoperoxidase, tyrosinase, prostaglandin H synthase (PHS), or 3-methylcholanthrene-induced rat liver microsomes. Starting with the quinones, the levels of depurinating adducts formed from E2-3,4-Q were much higher than that of the depurinating adduct from E2-2,3-Q. Similar results were obtained with lactoperoxidase or tyrosinase-catalyzed oxidation of 4-OHE2 and 2-OHE2, whereas with activation by PHS or microsomes, a relatively higher amount of the depurinating adduct from E2-2,3-Q was detected. These results demonstrate that the E2-3,4-Q is much more reactive with DNA than E2-2,3-Q. The relative reactivities of E2-3,4-Q and E2-2,3-Q to form depurinating adducts correlate with the carcinogenicity, mutagenicity, and cell-transforming activity of their precursors, the catechol estrogens 4-OHE2 and 2-OHE2. This is essential information for understanding the cancer risk posed by oxidation of the two catechol estrogens.

Topics: Adenine; Carcinogens; Deoxyadenosines; DNA; DNA Adducts; Estradiol; Estrogens, Catechol; Guanosine; In Vitro Techniques; Lactoperoxidase; Monophenol Monooxygenase; Mutagens; Oxidation-Reduction; Time Factors