2-hydroxyestrone and 4-hydroxyestrone

Diindolylmethane (DIM), a bioactive metabolite of indole-3-carbinol found in cruciferous vegetables, has proposed cancer chemoprevention activity in the breast. There is limited evidence of clinically relevant activity of DIM or long-term safety data of its regular use. A randomized, double-blind, placebo-controlled trial was conducted to determine the activity and safety of combined use of BioResponse DIM® (BR-DIM) with tamoxifen.. Women prescribed tamoxifen (n = 130) were randomly assigned oral BR-DIM at 150 mg twice daily or placebo, for 12 months. The primary study endpoint was change in urinary 2/16α-hydroxyestrone (2/16α-OHE1) ratio. Changes in 4-hydroxyestrone (4-OHE1), serum estrogens, sex hormone-binding globulin (SHBG), breast density, and tamoxifen metabolites were assessed.. Ninety-eight women (51 placebo, 47 DIM) completed intervention; compliance with treatment was >91%. BR-DIM increased the 2/16α-OHE1 ratio (+3.2 [0.8, 8.4]) compared to placebo (-0.7 [-1.7, 0.8], P < 0.001). Serum SHBG increased with BR-DIM compared to placebo (+25 ± 22 and +1.1 ± 19 nmol/L, respectively). No change in breast density measured by mammography or by MRI was observed. Plasma tamoxifen metabolites (endoxifen, 4-OH tamoxifen, and N-desmethyl-tamoxifen) were reduced in women receiving BR-DIM versus placebo (P < 0.001). Minimal adverse events were reported and did not differ by treatment arm.. In patients taking tamoxifen for breast cancer, daily BR-DIM promoted favorable changes in estrogen metabolism and circulating levels of SHBG. Further research is warranted to determine whether BR-DIM associated decreases in tamoxifen metabolites, including effects on endoxifen levels, attenuates the clinical benefit of tamoxifen.. ClinicalTrials.gov NCT01391689.

Topics: Adult; Antineoplastic Combined Chemotherapy Protocols; Biomarkers, Tumor; Breast Neoplasms; Double-Blind Method; Female; Humans; Hydroxyestrones; Indoles; Mammography; Middle Aged; Sex Hormone-Binding Globulin; Tamoxifen; Time Factors; Treatment Outcome

Higher levels of circulating estrogens and estrogen metabolites (EMs) have been associated with higher breast cancer risk. In breast tissues, reduced levels of terminal duct lobular unit (TDLU) involution, as reflected by higher numbers of TDLUs and acini per TDLU, have also been linked to elevated breast cancer risk. However, it is unknown whether reduced TDLU involution mediates the risk associated with circulating EMs. In a cross-sectional analysis of 94 premenopausal and 92 postmenopausal women referred for clinical breast biopsy at an academic facility in Vermont, we examined the associations of 15 EMs, quantified using liquid chromatography-tandem mass spectrometry, with the number of TDLUs and acini count/TDLU using zero-inflated Poisson regression with a robust variance estimator and ordinal logistic regression models, respectively. All analyses were stratified by menopausal status and adjusted for potential confounders. Among premenopausal women, comparing the highest vs. the lowest tertiles, levels of unconjugated estradiol (risk ratio (RR) = 1.74, 95 % confidence interval (CI) = 1.06-2.87, p trend = 0.03), 2-hydroxyestrone (RR = 1.74, 95 % CI = 1.01-3.01, p trend = 0.04), and 4-hydroxyestrone (RR = 1.74, 95 % CI = 0.99-3.06, p trend = 0.04) were associated with significantly higher TDLU count. Among postmenopausal women, higher levels of estradiol (RR = 2.09, 95 % CI = 1.01-4.30, p trend = 0.04) and 16α-hydroxyestrone (RR = 2.27, 95 % CI = 1.29-3.99, p trend = 0.02) were significantly associated with higher TDLU count. Among postmenopausal women, higher levels of EMs, specifically conjugated estrone and 2- and 4-pathway catechols, were also associated with higher acini count/TDLU. Our data suggest that higher levels of serum EMs are generally associated with lower levels of TDLU involution.

Topics: Adult; Breast; Breast Neoplasms; Chromatography, Liquid; Cross-Sectional Studies; Estradiol; Female; Humans; Hydroxyestrones; Image-Guided Biopsy; Middle Aged; Postmenopause; Premenopause; Tandem Mass Spectrometry

A sensitive, precise and accurate stable isotope dilution high-performance liquid chromatography-electrospray ionization mass spectrometry method has been developed for measuring endogenous 2- and 4-hydroxyestrones, the main catechol estrogens in human urine. Compared to the published methods using gas chromatography-mass spectrometry, this approach simplifies sample preparation and increases the throughput of analysis. The unique part of our method is the use of a simple and rapid derivatization step that forms a hydrazone at the C-17 carbonyl group of catechol estrogens. This derivatization step has greatly enhanced method sensitivity as well as HPLC separability of 2- and 4-hydroxyestrones. Standard curves were linear over a 100-fold calibration range with correlation coefficients for the linear regression curves typically greater than 0.996. The lower limit of quantitation for each catechol estrogen is 1 ng per 10-ml urine sample, with an accuracy of 97-99% and overall precision, including the hydrolysis, extraction and derivatization steps, of 1-3% for samples prepared concurrently and 2-11% for samples prepared in several batches. This method is adequate for measuring the low endogenous levels of catechol estrogens in urine from postmenopausal women.

Topics: Adult; Chromatography, High Pressure Liquid; Female; Humans; Hydrolysis; Hydroxyestrones; Isotopes; Middle Aged; Reproducibility of Results; Sensitivity and Specificity; Spectrometry, Mass, Electrospray Ionization

Estrogen metabolism is altered in most, if not all, breast cancer tumors. These alterations primarily lead to the formation of the catechol estrogen metabolites, 2- and 4-hydroxyestrogens, which can generate superoxide anion radicals (O(2)(*)(-)) through the redox cycling of semiquinone/quinone derivatives. In breast cancer cells, the activity of nitric oxide synthase is also frequently elevated, resulting in an increased level of exposure to nitric oxide ((*)NO). Since (*)NO rapidly reacts with O(2)(*)(-) to produce the peroxynitrite anion (ONOO(-)), this study was undertaken to determine whether ONOO(-) can be generated when 2- and 4-hydroxyestrogens are incubated in vitro with (*)NO donor compounds. Using dihydrorhodamine 123 as a specific probe for ONOO(-) formation, a ratio of 100 microM dipropylenetriamine NONOate (DPTA/NO) to 10 microM 4-hydroxyestradiol (4-OHE(2)) gave an optimal ONOO(-) production of 11.9 +/- 1.9 microM (mean +/- SD). Quantification of ONOO(-) was not modified by mannitol, supporting the idea that the hydroxyl radical was not involved. This production of ONOO(-) required the presence of the catechol structure of estrogen metabolites since all methoxyestrogens that were tested were inactive. Hydroxyestrogen metabolites derived from estradiol showed the same efficiency in producing ONOO(-) as those originating from estrone. With DPTA/NO, the 4-hydroxyestrogens generated 30-40% more ONOO(-) than the 2-hydroxyestrogens. Optimal production of ONOO(-) was assessed with DPTA/NO and diethylenetriamine NONOate (initial (*)NO generation rates of 0.76 and 0.08 microM min(-1), respectively). With faster (*)NO-releasing compounds, such as diethylamine NONOate and spermine NONOate, lower levels of ONOO(-) were detected. These data suggest that once the optimal concentration of (*)NO was obtained, the reaction between (*)NO and 4-OHE(2) was saturated. The excess of (*)NO would probably react with aqueous oxygen to form nitrite (NO(2)(-)). Since the third-order reaction rate for the reaction between 2(*)NO and O(2) is 2 x 10(6) M(-2) s(-1), it can therefore be suggested that the reaction between (*)NO and 4-OHE(2) occurs at a faster rate.

Topics: Chromatography, High Pressure Liquid; Estradiol; Estrogens, Catechol; Hydroxyestrones; Mass Spectrometry; Nitrates; Nitric Oxide

Catechol estrogens are considered critical intermediates in estrogen-induced carcinogenesis. We demonstrated previously that 17beta-estradiol (E(2)), estrone (E(1)) and four of their catechol estrogens, 2- and 4-hydroxyestradiols (2- and 4-OHE(2)), and 2- and 4-hydroxyestrones (2- and 4-OHE(1)) induce morphological transformation in Syrian hamster embryo (SHE) fibroblasts, and the transforming abilities vary as follows: 4-OHE(1) > 2-OHE(1) > 4-OHE(2) > 2-OHE(2) vertical line E(2), E(1). To examine the involvement of catechol estrogens in the initiation of hormonal carcinogenesis, we studied the ability of E(2), E(1) and their catechol estrogens to induce DNA adducts in SHE cells by using a (32)P-post-labeling assay. DNA adducts were detected in cells treated with each of all the catechol estrogens at concentrations of 10 microg/ml for 1 h and more. 2- or 4-OHE(2) formed a single DNA adduct, which was chromatographically distinct from each other. In contrast, 2- or 4-OHE(1) produced one major and one minor adduct, and the two adducts formed by each catechol estrogen exhibited identical mobilities on the chromatograms. Neither E(2) nor E(1) at concentrations up to 30 microg/ml induced DNA adducts. The abilities of the estrogens to induce DNA adducts were ranked as follows: 4-OHE(1) > 2-OHE(1) > 4-OHE(2) > 2-OHE(2) > > E(2), E(1), which corresponds well to the transforming and carcinogenic abilities of the estrogens. In addition, the level of DNA adducts induced by the catechol estrogens was markedly decreased by co-treatment of cells with the antioxidant L-ascorbic acid. The results indicate the possible involvement of oxidative metabolites of catechol estrogens of E(2) and E(1) in the initiation of endogenous estrogen-induced carcinogenesis.

Topics: Animals; Antioxidants; Ascorbic Acid; Cell Survival; Cell Transformation, Neoplastic; Cricetinae; DNA Adducts; Estradiol; Estrogens, Catechol; Fibroblasts; Hydroxyestrones; Mesocricetus

Topics: Animals; Carcinogens; Estrogens; Female; Humans; Hydroxyestrones; Mutagens; Neoplasms

Catechol estrogens are major estrogen metabolites in mammals and are the most potent naturally occurring inhibitors of catecholamine metabolism. These estrogen compounds have been implicated in carcinogenic activity and the 4/2-hydroxyestradiol concentration has been shown to be elevated in neoplastic human mammary tissue compared to normal human breast tissue. Three human liver UDP-glucuronosyltransferases, UGT2B7, UGT1A1, and UGT1A3, have been shown to catalyze the glucuronidation of catechol estrogens and lead to their enhanced elimination via urine or bile. The present study was designed to study the kinetic interaction of expressed human UGT2B7(Y) or (H), UGT1A1, and UGT1A3 toward 2- and 4-hydroxycatechol estrogens. cDNAs encoding UGT2B7(Y) or (H), UGT1A1, and UGT1A3 were expressed in HK293 cells, and cell homogenates or membrane preparations were used to determine their glucuronidation ability. UGT2B7(Y) reacted with higher efficiency toward 4-hydroxyestrogenic catechols, whereas UGT1A1 and UGT1A3 showed higher activities toward 2-hydroxyestrogens. UGT2B7(H) catalyzed estrogen catechol glucuronidation with efficiencies similar to UGT2B7(Y). Flunitrazepam (FNZ), a competitive inhibitor of morphine glucuronidation in hepatic microsomes, competitively inhibited catechol estrogen glucuronidation catalyzed by UGT2B7(Y), UGT1A1, and UGT1A3. Buprenorphine, an opioid substrate that reacts at high efficiency with each of these UGTs, was also studied. FNZ competitively inhibited buprenorphine glucuronidation with UGT1A1 and UGT2B7 but had no inhibitory activity toward UGT1A3. This suggests that buprenorphine and 2-hydroxycatechol estrogens react with separate active sites of UGT1A3. A catecholamine, norepinephrine, did not inhibit UGT2B7(Y)-, UGT1A1-, and UGT1A3-catalyzed glucuronidation of catechol estrogens. These results also suggest that drug-endobiotic interactions are possible in humans and may have implication in carcinogenesis.

Topics: Estradiol; Estrogens, Catechol; Flunitrazepam; Glucuronates; Glucuronosyltransferase; Humans; Hydroxyestrones; Isoenzymes; Norepinephrine

The synthesis of N-acetylcysteine conjugates of 2-hydroxyestrone (2-OHE1) and 4-hydroxyestrone (4-OHE1) is described. The reaction of estrone 2,3-quinone with N-acetylcysteine provided 2-OHE1 and its C-4 and C-1 thioether conjugates in a ratio of 1:1, while estrone 3,4-quinone with N-acetylcysteine gave 4-OHE1 and its C-2 thioether conjugate as a sole product. Their structures were characterized by inspection of NMR spectra, chemical derivatization (methylation and acetylation), and comparison with the reactivity of 4-bromoestrone 2,3-quinone or 2-bromoestrone 3,4-quinone toward N-acetylcysteine.

Topics: Acetylcysteine; Estrogens, Catechol; Hydroxyestrones; Magnetic Resonance Spectroscopy; Molecular Structure

As part of an ongoing investigation of the role of metabolic activation of estrogens in the genesis of cancers such as estrogen-induced renal tumors in hamsters, we have 1) determined steroid-17 beta-oxidoreductase activity of microsomes and cytosol prepared from hamster kidney and liver; 2) compared the rates of 2-, 4-, and 16 alpha-hydroxylations of estrone by microsomes from hamster kidney and liver; and 3) determined the rates of inactivation of 2- and 4-hydroxyestrone by catechol-O-methyltransferase from hamster kidney and by purified enzyme. Microsomal steroid-17 beta-oxidoreductase activity in hamster kidney and liver was low and favored the conversion of estrone to estradiol. Cytosolic steroid-17 beta-oxidoreductase activity was only barely detectable in both liver and kidney. Using hepatic microsomes, the rate of 2-hydroxylation of estrone was comparable to that found previously using estradiol as substrate, whereas 4-hydroxylation of estrone was double that of estradiol. Using renal microsomes, the rates of 2- and 4-hydroxylation of estrone were 10- to 20-fold higher than those with estradiol as substrate, and the ratio of 2- to 4-hydroxylation was about 2:1. Fadrozole hydrochloride was an equally good inhibitor of rates of 2- and 4-hydroxylation of estrone (20 microM) by hepatic microsomes (IC50, approximately 25 microM). Corresponding IC50 values with renal microsomes were less than 2 microM, and 2-hydroxylation of estrone was inhibited by Fadrozole hydrochloride up to 15% more than 4-hydroxylation. Treatment of hamsters with estradiol for 2 months decreased rates of 2- and 4-hydroxylation of estrone by renal microsomes by approximately 95%. The rate of conversion of estrone to 16 alpha-hydroxyestrone by hepatic microsomes was 10-20% that of 2-hydroxylation. Renal microsomes catalyzed 16 alpha-hydroxylation of estrone at an even lower rate (approximately 5% of that of 2-hydroxylation). Rates of O-methylation of 2- and 4-hydroxyestrone by hamster kidney cytosol were comparable to those of 2- and 4-hydroxyestradiol. In conclusion, conversion of estrone to its catechol metabolites by microsomes of hamster kidney, a target organ of estrogen-induced carcinogenesis, is quantitatively more important than the conversion to 16 alpha-hydroxyestrone. The findings are consistent with the postulated role of catechol estrogens generated in situ in estrone-induced carcinogenesis.

Topics: Animals; Catechol O-Methyltransferase; Cell Transformation, Neoplastic; Chromatography, Gas; Cricetinae; Estrogens; Estrone; Hydroxyestrones; Hydroxylation; Kidney; Male; Mesocricetus; Microsomes; Microsomes, Liver; Steroid 16-alpha-Hydroxylase

In vitro metabolic conjugation of the catechol estrogens, 2-hydroxyestrone and 4-hydroxyestrone, has been investigated by means of HPLC with electrochemical detection. Sulfation of 2-hydroxyestrone and 4-hydroxyestrone with the rat liver 105 000 g supernatant fortified with 3'-phosphoadenosine-5'-phosphosulfate provided the 2- and 4-monosulfates, respectively. Glucuronidation of the two catechols with the rat and human liver 1500 g supernatant in the presence of uridine-5'- phosphoglucuronic acid gave the 2- and 4-glucuronides, respectively. In contrast, incubation with the guinea pig liver 1500 g supernatant yielded both isomeric monoglucuronides . When 2'-hydroxyestrone was incubated with rat liver 1500 g supernatant and S-adenosyl-L-methionine, the 2- and 3-monomethyl ethers were formed in an equal amount, while 4-hydroxyestrone was transformed into the 4-methyl ether in 12 times greater yield than the 3-methyl ether. The participation of sulfation and glucuronidation in the formation of guaiacol estrogens is discussed.

Topics: Animals; Chromatography, High Pressure Liquid; Electrochemistry; Estrogens, Catechol; Estrogens, Conjugated (USP); Estrone; Female; Glucuronates; Guaiacol; Guinea Pigs; Humans; Hydroxyestrones; In Vitro Techniques; Liver; Male; Rats; Rats, Inbred Strains; Sulfates

The mechanisms underlying the differences in uterotrophic potency between 2- and 4-hydroxyestrogens were explored. Doses of estradiol (E2)(10 micrograms/kg), 2-OHE2 (500 micrograms/kg) and 4-OHE2 (100 micrograms/kg) sufficient to induce near maximal cell nuclear estrogen receptor (ERn) binding were injected subcutaneously into 26 day old female rats. Uterine ERn concentrations declined more rapidly after 2-OHE2 than after E2 or 4-OHE2. E2 and 4-OHE2 both elicited a significant increase in uterine wet weight, measured at 24-36 hrs after injection. 2-OHE2 had no significant effect and neither synergized with nor antagonized the effects of simultaneously administered E2 or 4-OHE2. Under in vitro conditions at 25 degrees C, 2-hydroxyestrone (2-OHE1) and 2-OHE2 both dissociated from the receptors more rapidly than either their parent monophenolic estrogens or the corresponding 4-hydroxyestrogens. These results suggest that differences in estrogenic potency between 2- and 4-hydroxyestrogens may partly be a function of the dissociation kinetics of their estrogen receptor complexes.

Topics: Animals; Binding, Competitive; Cell Nucleus; Cytosol; Estradiol; Estrone; Female; Hydroxyestrones; Kinetics; Rats; Rats, Inbred Strains; Receptors, Estrogen; Uterus

A high performance liquid chromatographic method is described for the rapid, non-destructive separation of a number of physiologically important steroidal estrogens, including the labile catechol estrogens. This procedures uses a "Diol" column and gradient elution to separate in a single run, estrogens ranging from 2-methoxy estrone, one of the least polar C18 steroids, to estriol, one of the most polar. Simpler, isocratic conditions, are provided for the separation of estrogens of similar polarity. A semi-preparative column of similar composition was used for the purification of samples containing 25 to 50 mg of individual steroids.

Topics: Chromatography, High Pressure Liquid; Estradiol; Estrogens; Estrogens, Catechol; Hydroxyestrones

The synthesis of [2H8] estradiol, [2H7] estrone, [2H6] 2-hydroxyestrone and [2H6] 4-hydroxyestrone from estrone (as a source) is described. The high isotopical purity renders the labelled compounds as suitable carriers and internal standards for quantitative gas chromatography - mass spectrometry. The content of protonium-form (i.e. natural) estrogens in the labelled derivatives ranged from 0.12% to 2.58%. The performance of these compounds in quantitative assays using selected ion monitoring has been established; and this allows the determination of estrogens from biological material in the lower picogram range.

Topics: Deuterium; Estradiol; Estrone; Gas Chromatography-Mass Spectrometry; Hydroxyestrones; Isotope Labeling; Reference Standards

Topics: Animals; Catechols; Estrogens; Estrogens, Catechol; Feedback; Female; Gonadotropins, Pituitary; Humans; Hydroxyestrones; Hypothalamo-Hypophyseal System; Menstruation; Ovary; Rats

Topics: 6-Ketoprostaglandin F1 alpha; Animals; Estradiol; Estrogens, Catechol; Estrone; Female; Humans; Hydroxyestrones; Prostaglandins; Prostaglandins E; Prostaglandins F; Rats; Uterus

A method for the determination of 2- and 4- hydroxylated estrone and estradiol in pregnancy urine by high-performance liquid chromatography with electrochemical detection (HPLC-ECD) is described. The urine catechol estrogens were deconjugated, purified by adsorption on alumina, and subjected to HPLC-ECD. Two pairs of isomeric catechol estrogens were distinctly separated on a microBondapak c18 column with acetonitrile-0.5% ammonium dihydrogen phosphate (pH 3.0). The amounts of these four compounds were satisfactorily determined with a quantitation limit of 1 ng using 4-hydroxy-16 oxoestradiol 17 acetate as an internal standard. The validity of the present methods for the determination of urine catechol estrogens was verified by the recovery test.

Topics: Chromatography, High Pressure Liquid; Estradiol; Estrone; Female; Humans; Hydroxyestrones; Isomerism; Pregnancy; Reference Values

Topics: Animals; Castration; Dose-Response Relationship, Drug; Estradiol; Estrone; Female; Follicle Stimulating Hormone; Hydroxyestrones; In Vitro Techniques; Luteinizing Hormone; Pituitary Gland; Receptors, Estrogen; Sheep

When [6,7-3H]estradiol was incubated with tissue homogenates of the brain, the pituitary, and the liver of two human female fetuses, a number of radioactive metabolites more "polar" than the incubated substrate were detected. Among these, the identification of two types of catecholestrogens, i.e. the 2- and 4-hydroxyestrogens, was of major interest. Compared on the basis of wet weight of tissues (250 mg), the conversion of estradiol to 2-hydroxyestrogens (2-hydroxyestradiol and 2-hydroxyestrone) was 0.8% in the frontal cortex, 1.0% in the hypothalamus, 2.1% in the pituitary, and 7.8% in the liver. For the first time, the formation of 4-hydroxyestrogens was demonstrated. The percentages of incubated estradiol hydroxylated at C-atom 4 (4-hydroxyestradiol and 4-hydroxyestrone) were 0.5 in the cortex, 0.4% in the hypothalamus, .1% in the pituitary, and 0.5% in the liver. The results show that fetal brain and pituitary tissue can hydroxylate estradiol in positions 2 and 4 to a similar extent, whereas in the liver, about 15 times more 2-hydroxy than 4-hydroxy compounds are formed. Moreover, the 2-hydroxylating capacity of the liver is definitely greater than that of the brain, whereas the 4-hydroxylating capacity is about the same as that of the brain.

Topics: Brain; Estradiol; Estrogens, Catechol; Female; Humans; Hydroxyestrones; Hydroxylation; Liver; Pituitary Gland