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PubMed1987

Iodoacetic acid and related sulfhydryl reagents fail to inhibit cell-cell communication: mechanisms of immunotoxicity in vitro.

The present study was undertaken to examine the effects of iodoacetic acid, a non-phorbol tumor promoter, on metabolic cooperation between mutant human fibroblasts as measured by [14C]citrulline incorporation. Other thiol-reactive polyphenolic compounds such as hydroquinone and 2-hydroxyestrone were also examined. 12-O-Tetradecanoyl phorbol-13-acetate (TPA), a potent skin tumor promoter, inhibited the cell-cell communication by more than 60% at 20 ng/ml. However, iodoacetic acid, hydroquinone, and 2-hydroxyestrone, had no effect on the process even at cytotoxic concentrations. Induction of intercellular contact (agglutination) among lymphocytes during the course of phytohemagglutinin (PHA)-induced blastogenesis was monitored turbidometrically at 620 nm. Hydroquinone and 2-hydroxyestrone suppressed the PHA-induced lymphocyte agglutination at 1-2 microM in vitro concentrations while iodoacetic acid was devoid of any effects at concentrations up to 100 microM. Hydroquinone and 2-hydroxyestrone concomitantly suppressed PHA-induced lymphocyte blastogenesis at 1-2 microM in vitro concentrations while the suppression by iodoacetic acid was significant at 10 microM. All 3 compounds failed to disrupt microtubule assembly, a sulfhydryl-dependent process, in a rat brain crude extract. However, p-benzoquinone, an oxidation product of hydroquinone, did inhibit the process at 1 mM. In summary, these studies suggest that, unlike TPA, thiol-reactive non-phorbol tumor promoters and polyphenolic compounds do not inhibit cell-cell communication between mutant human fibroblasts. Although the compounds demonstrate diverse molecular mechanisms of action, they all inhibit in vitro immune functions suggesting that immunosuppression may play a role in tumor promotion.

Agglutination↗
PubMed2006

A hormonal association between estrogen metabolism and proliferative thyroid disease.

OBJECTIVE: To illustrate a relationship between proliferative thyroid disease and estrogen metabolism through the analysis of urinary estrogen metabolites. STUDY DESIGN AND SETTING: Case-control study of 49 subjects with proliferative thyroid disorders and matching them to 49 controls. Urinary estrogen metabolite ratios were obtained, measuring 2-hydroxyestrone, an anti-proliferative metabolite, to 16alpha-hydroxyestrone, a proliferative metabolite. The patients were stratified into low (0 to 1.00), medium (1.01 to 2.00), or high (>2.00) groups according to their estrogen metabolite ratio. RESULTS: Fifty-one percent (25 of 49) of the cases had a low 2/16 ratio compared to 31% (15 of 49) in the control group while 20% (10 of 49) of the control group had a high 2/16 ratio as compared to 8% (4 of 49) in the case group (P value < 0.05). CONCLUSIONS: Increased 16alpha-hydroxyestrone activity compared to 2-hydroxyestrone activity appears to be associated with proliferative thyroid disease. SIGNIFICANCE: Further study of estrogen metabolites in relation to proliferative thyroid disease is warranted and may lead to implications for new treatment modalities for proliferative thyroid disease. EBM RATING: B-3b.

Adolescent↗
PubMed2003

Influence of postmenopausal hormone replacement therapy on an estrogen metabolite biomarker of risk for breast cancer.

Whether postmenopausal hormone-replacement therapy (HRT) increases the risk of breast cancer remains controversial, despite numerous epidemiological studies. We approached the question from a biochemical rather than an epidemiological direction - we hypothesized that if estrogen administration increases the risk of breast cancer, it should also alter a known estrogen biomarker of risk towards what has been observed in patients who already have breast cancer. The specific biomarker we studied was the ratio of the urinary excretion of two principal estradiol metabolites, 2-hydroxyestrone and 16 alpha-hydroxyestrone, which is markedly decreased in women with breast cancer and women with familial risk for breast cancer. We studied 34 healthy postmenopausal women not on HRT and 19 women on HRT (Premarin 0.625 mg daily plus Provera, 2.5 mg daily, in women with a uterus and Premarin alone in women without a uterus); treatment duration ranged from 3 months to 15 years. We also studied four women with recently diagnosed, untreated breast cancer. The women with breast cancer showed a significantly lower 2-hydroxyestrone to 16 alpha-hydroxyestrone ratio than control women on HRT (1.35 +/- 0.13 vs. 2.71 +/- 0.84; p < 0.0001). There was no significant difference in the metabolite ratio between healthy women on HRT and women not on HRT (2.82 +/- 0.92 vs. 2.71 +/- 0.84). There was no significant difference between women receiving Premarin alone and women receiving Premarin plus Provera (2.46 +/- 0.84 vs. 3.13 +/- 0.90), and neither differed significantly from women not on HRT (2.71 +/- 0.84). The finding that the ratio of women on HRT was not decreased to or toward the ratio in women with breast cancer can be interpreted, we believe, as a suggestive item of biochemical evidence that HRT is not a risk for breast cancer.

Biomarkers, Tumor↗
PubMed2003

[Estrogen-dependent neoplasia - what is the significance of estradiol metabolites].

Estradiol can be metabolized to substances eliciting different, partly opposite effects even at low concentrations as shown in own investigations, e. g., regarding (anti)angiogenic actions. Specific anticancerogenic effects are ascribed to 2-hydroxyestrone and particularly 2-methoxyestradiol. In contrast, 16alpha-hydroxyestrone and the 4-hydroxyestrogens may be genotoxic under certain circumstances. Furthermore there are indications that endogenous production of proliferation-stimulating metabolites is raised in some cancers. Especially the urinary excretion of 2-hydroxyestrone to 16alpha-hydroxyestrone was investigated showing in own and other clinical studies a lower ratio in postmenopausal women with breast cancer. Research is ongoing inasfar the determination of estradiol metabolites also in blood or directly in the breast tissue by means of sensitive laboratory methods may allow predictive statements. However, it has be to consider that estradiol metabolism can be influenced by external factors such as nutrition, smoking, sports and drugs such as L-thyroxine and H2-antagonists. We were able to demonstrate that estradiol metabolism during estradiol treatment depends on the application mode and might be differently influenced by addition of the various progestins. Whether the investigation of gene polymorphism of enzymes, which are involved in estradiol metabolism, may be helpful for the assessment or treatment of risk patients, to our opinion needs further research.

2-Methoxyestradiol↗
PubMedDate not supplied

A randomized phase II trial of indole-3-carbinol in the treatment of vulvar intraepithelial neoplasia.

The aim of this study was to determine the potential therapeutic benefits of indole-3-carbinol (I3C) in the management of vulvar intraepithelial neoplasia (VIN). Women with histologically confirmed high-grade VIN were randomized to receive 200 and 400 mg/day of I3C. Symptomatology by visual analog scale and vulvoscopic appearance were assessed at recruitment, 6 weeks, 3 months, and 6 months. Tissue biopsy to determine histologic response was obtained at completion of the study period. Urine samples were obtained at each visit to determine 2-hydroxyestrone to 16-alpha-hydroxyestrone ratios. Data from 12 women were suitable for analysis. There was a significant improvement in symptomatology with the introduction of I3C (itch, P= 0.018; pain, P= 0.028). Lesion size and severity were also significantly reduced (size, P= 0.005; appearance, P= 0.046). In addition, there was a significant increase in 2-hydroxyestrone to 16-alpha-hydroxyestrone ratio following commencement of I3C, P= 0.05. However, tissue biopsy from the worst-affected vulval areas revealed no improvement in grade of VIN during the 6-month period, P= 0.317. There were no significant differences in results between those women taking 200 mg/day of I3C and those on 400 mg/day. This study has shown significant clinical improvement in symptomatology and vulvoscopic appearance of VIN with I3C therapy. Further clinical and scientific investigations are required to support these preliminary findings.

Adult↗
PubMed1977

Direction of estradiol metabolism as a control of its hormonal action--uterotrophic activity of estradiol metabolites.

The uterotrophic activities of the catechol metabolites of estradiol 2-hydroxyestrone, 2-methoxyestrone and 2-hydroxyestradiol were measured under conditions of continuous administration of sc implanted paraffin pellets. The activity of these estrogens was compared to that of estradiol-17beta and its other principal metabolites estrone, estriol and 15alpha-hydroxyestriol (estetrol). The major catechol estrogens, 2-hydroxyestrone and 2-methoxyestrone, and the pregnancy metabolite, 15alpha-hydroxyestriol, exhibited no uterotrophic activity. The minor catecholestrogen, 2-hydroxyestradiol, showed some activity whose character was different from that exhibited by implants of estradiol, estrone and estriol all of which were equipotent uterotrophic agents. Implants of 2-hydroxyestrone in the presence of estradiol or estriol pellets did not diminish the response to the latter indicating that the 2-hydroxyestrone is not antiestrogenic under these conditions. It is concluded that the direction of estradiol metabolism can have a profound influence on the expression of peripheral hormonal activity with hydroxylation at C-2 terminating and hydroxylation at C-16 extending it.

Animals↗
PubMed2000

Brassica vegetable consumption shifts estrogen metabolism in healthy postmenopausal women.

Previous studies suggest that the estrogen metabolite 16alpha-hydroxyestrone acts as a breast tumor promoter. The alternative product of estrogen metabolism, 2-hydroxyestrone, does not exhibit estrogenic properties in breast tissue, and lower values of the ratio 2-hydroxyestrone:16alpha-hydroxyestrone (2:16) in urine may be an endocrine biomarker for greater breast cancer risk. Vegetables of the Brassica genus, such as broccoli, contain a phytochemical, which may shift estrogen metabolism and increase the 2:16 ratio. Adding 500 g/day of broccoli to a standard diet shifts 2:16 values upward in humans; however, it is unknown as to whether healthy women are able to consume a sufficient quantity of Brassica to affect breast cancer risk through this mechanism. In this study, 34 healthy postmenopausal women participated in an intensive intervention designed to facilitate the addition of Brassica to the daily diet. The diet was measured by repeated 24-h recall, and estrogen metabolites were measured by enzyme immunoassay in 24-h urine samples. In a crude analysis, there was a nonsignificant increase in the urinary 2:16 ratio associated with greater Brassica consumption. With adjustment for other dietary parameters, Brassica vegetable consumption was associated with a statistically significant increase in 2:16 values, such that for each 10-g/day increase in Brassica consumption, there was an increase in the 2:16 ratio of 0.08 (95% confidence interval, 0.02-0.15). To the extent that the 2:16 ratio, as measured in urine, is associated with breast cancer risk, future research should consider Brassica vegetable consumption as a potentially effective and acceptable dietary strategy to prevent breast cancer.

Aged↗
PubMed1989

Separation of urinary metabolites of radiolabelled estrogens in man by HPLC.

A method to separate radiolabelled urinary estrogens by high performance liquid chromatography (HPLC) is described. Estrogen glucuronides were isolated from the urine of women receiving bolus injections of [4-14C]estrone or [4-14C]estradiol by adsorption on Sep-Pak C18 cartridges and subsequent DEAE Sephadex A25 column chromatography. Following enzyme hydrolysis, free estrogens were extracted and concentrated in methanol-water containing ascorbic acid. HPLC was performed either by C18 reversed phase chromatography using different concentrations of acetonitrile with or without tetrahydrofurane in phosphate buffer or methanol-water as mobile phases, or on a Diol column using chloroform-isooctane-n-hexane or isopropanol-isooctane-n-hexane as mobile phases. 3H-labelled estrogens were added as internal standards, and urinary [14C]estriol, [14C]estradiol and [14C]estrone concentrations could be measured with an interassay coefficient of variation less than 5%. Interassay coefficients of variation for [14C]2-hydroxyestriol, [14C]16 alpha-hydroxyestrone, [14C]2-hydroxyestradiol, [14C]2-hydroxyestrone and [14C]2-methoxyestrone were between 5 and 10%, while interassay coefficients of variation for [14C]4-hydroxyestrone was 14.6%. Recovery of the unstable catechol estrogen 2-hydroxyestrone was comparable to the recovery of the other estrogen metabolites, due to the addition of ascorbic acid throughout the different pre-chromatographic steps. Our method is suitable for the separation of the major labelled estrogen metabolites found in human urine following administration of radiolabelled estrone or estradiol.

Aminoglutethimide↗
PubMed1976

Metabolism of radioactive 17 beta-estradiol 3-methyl ether by humans.

A mixture of 2-3H and 4-14C-17beta-estradiol 3-methyl ether was administered orally to a man and to a woman. 34 and 35 percent of the 3H was liberated into the body water of the man and of the woman, respectively, reflecting reactions involving position 2. The metabolism of estradiol methyl ether was qualitatively similar to that observed previously for radioactive estradiol administered intravenously to the same subjects, as judged by the measurement of various urinary metabolites by reverse isotope dilution. Evidence was obtained for hydroxylation at position 2 without demethylation by the isolation of urinary 2-hydroxyestrone 3-methyl ether which retained 33% of the original 3H. This 3H was presumably at position 1, resulted from an NIH shift which does not occur during hydroxylation of estrone or estradiol. This was confirmed by subsequent administration of a mixture of 4-14C and 3H-(methoxyl)-estradiol 3-methyl ether to the man. There was no evidence (by reverse isotope dilution) for 1-hydroxyestrone, 1-hydroxyestrone 3-methyl ether, 4-hydroxyestrone 3-methyl ether or 4-hydroxyestradiol 3-methyl ether as urinary metabolites of estradiol 3-methyl ether.

Adult↗
PubMed2006

Analysis of fifteen estrogen metabolites using packed column supercritical fluid chromatography-mass spectrometry.

Packed column supercritical fluid chromatography with tandem mass spectrometry was used for the separation of estrone, estradiol, estriol, 16-epiestriol, 17-epiestriol, 16-ketoestradiol, 16alpha-hydroxyestrone, 2-methoxyestrone, 4-methoxyestrone, 2-hydroxyestrone-3-methyl ether, 2-methoxyestradiol, 4-methoxyestradiol, 2-hydroxyestrone, 4-hydroxyestrone, and 2-hydroxyestradiol. A gradient of methanol in carbon dioxide (0-30% methanol in 15 min, 2% change/min) at a flow rate of 2 mL/min and cyanopropyl silica column connected in series with a diol column, both 2.1 mm i.d. x 150 mm long, packed with 5-mum spherical silica-based particles, resulted in the separation and quantification of all 15 estrogens in less than 10 min. The limit of detection (LOD) and limit of quantitation (LOQ) of this pSFC MS/MS method was determined to be 0.5 (S/N = 3), and 5 pg, respectively. Compared with RP-HPLC MS analysis of the same mixture in terms of speed of analysis and sensitivity, pSFC MS is much faster, 10 versus 70 min, with comparable LOD and LOQ.

Carbon Dioxide↗
PubMed1996

p-Quinone methides are the major decomposition products of catechol estrogen o-quinones.

The mechanism of catechol estrogen-induced carcinogenesis could involve alkylation of critical cellular macromolecules by electrophilic quinoids. The o-quinones formed from peroxidase/P450-catalyzed oxidation of catechol estrogens have previously been implicated as the ultimate carcinogens. In the present study, we have shown that additional reactive intermediates can be produced from isomerization of the catechol estrogen o-quinones to highly electrophilic p-quinone methides (QMs). The o-quinones of the catechol estrogens were incubated at 37 degrees C (pH 7.4) in the absence of GSH. Aliquots were removed at various times and combined with GSH. The GSH adducts were isolated and characterized by 1H-NMR, UV, and electrospray mass spectrometry. The o-quinone of 2-hydroxyestrone isomerized to two QMs; a QM stabilized by one alkyl substituent in the B ring, 2-OHE-QM1 (3-hydroxy-1-(10),3(4),5(6)-oestratrien-2,17-dione) and one having two alkyl substituents on the methylene group in the C ring, 2-OHE-QM2 (2-hydroxy-1(2),4(5),9(10)-oestratrien-3,17-dione). Only one QM was observed from the o-quinone of 4-hydroxyestrone, 4-OHE-QM2 (4-hydroxy-1(2),4(5),9(10)- oestratrien-3,17-dione) which is analogous to the C ring analog (2-OHE-QM2) from the o-quinone of 2-hydroxyestrone. The GSH adduct of 4-OHE-QM2 decomposed at pH 7.4 to give 9(11)-dehydro-4-hydroxyestrone as the major product. Finally, the disappearance of the estrogen o-quinone GSH adducts correlated with the formation of the GSH conjugates of the QMs. These data suggest that in cells with low levels of GSH, the formation of these potent electrophiles represents the major reaction pathway for estrogen o-quinones. The implications of the o-quinone/QM pathway for the in vivo effects of catechol estrogens are not known; however, given the direct link between excessive exposure to endogenous estrogens and the enhanced risk of breast cancer, the potential for formation of additional reactive intermediates needs to be explored.

Estrogens, Catechol↗
PubMed1999

Multifunctional aspects of the action of indole-3-carbinol as an antitumor agent.

Previous studies from this laboratory have suggested that 2-hydroxyestrone is protective against breast cancer, whereas the other principal metabolite, 16 alpha-hydroxyestrone, and the lesser metabolite quantitatively, 4-hydroxyestrone, are potent carcinogens. Attempts to directly decrease the formation of the 16-hydroxylated metabolite were either unsuccessful or required such high levels of the therapeutic agent as to be impractical. On the other hand the concentration of the protective metabolite, 2-hydroxyestrone, proved to be readily modulated by a variety of agents, both in the direction of increased protection and the opposite direction, increased risk by a variety of agents and activities. We have focussed our attention on indole-3-carbinol, a compound found in cruciferous vegetables, and its further metabolites in the body, diindolylmethane (DIM) and indolylcarbazole (ICZ), because of its relative safety and multifaceted activities. It has been shown that it induces CyP4501A1, increasing 2-hydroxylation of estrogens, leading to the protective 2-OHE1, and also decreases CyP1B1 sharply, inhibiting 4-hydroxylation of estradiol, thereby decreasing the formation of the carcinogenic 4-OHE1. In addition to these indirect effects as a result of altered estrogen metabolism, indole-3-carbinol has been shown to have direct effects on apoptosis and cyclin D, resulting in blockage of the cell cycle. In addition to its antitumor activity in animals, it has also been shown to be effective against HPV-mediated tumors in human patients. All of these responses make the study of its behavior as a therapeutic agent of considerable interest.

Anticarcinogenic Agents↗
PubMed2002

Estrogen metabolism and the diet-cancer connection: rationale for assessing the ratio of urinary hydroxylated estrogen metabolites.

Estrogens are known for their proliferative effects on estrogen-sensitive tissues resulting in tumorigenesis. Results of experiments in multiple laboratories over the last 20 years have shown that a large part of the cancer-inducing effect of estrogen involves the formation of agonistic metabolites of estrogen, especially 16-alpha-hydroxyestrone. Other metabolites, such as 2-hydroxyestrone and 2-hydroxyestradiol, offer protection against the estrogen-agonist effects of 16-alpha-hydroxyestrone. An ELISA method for measuring 2- and 16-alpha-hydroxylated estrogen (OHE) metabolites in urine is available and the ratio of urinary 2-OHE/16-alpha-OHE (2/16-alpha ratio) is a useful biomarker for estrogen-related cancer risk. The CYP1A1 enzyme that catalyzes 2-hydroxyestrone (2-OHE1) formation is inducible by dietary modification and supplementation with the active components of cruciferous vegetables, indole-3-carbinol (I-3-C), or diindolylmethane (DIM). Other dietary components, especially omega-3 polyunsaturated fatty acids and lignans in foods like flax seed, also exert favorable effects on estrogen metabolism. Thus, there appear to be effective dietary means for reducing cancer risk by improving estrogen metabolism. This review presents the accumulated evidence to help clinicians evaluate the merit of using tests that measure estrogen metabolites and using interventions to modify estrogen metabolism.

Breast Neoplasms↗
PubMed2000

Involvement of genotoxic effects in the initiation of estrogen-induced cellular transformation: studies using Syrian hamster embryo cells treated with 17beta-estradiol and eight of its metabolites.

To examine a direct involvement of genotoxic effects of estrogens in the initiation of hormonal carcinogenesis, the abilities of 17beta-estradiol (E2) and 8 of its metabolites to induce cellular transformation and genetic effects were studied using the Syrian hamster embryo (SHE) cell model. Treatment with E2, estrone (E1), 2-hydroxyestrone (2-OHE1), 4-hydroxyestrone (4-OHE1), 2-methoxyestrone (2-MeOE1), 16alpha-hydroxyestrone (16alpha-OHE1), 2-hydroxyestradiol (2-OHE2), 4-hydroxyestradiol (4-OHE2) or estriol (E3) for I to 3 days inhibited SHE cell growth in a concentration-dependent manner. Concentration-dependent increases in the frequency of morphological transformation in SHE cells were exhibited by treatment for 48 hr with each of all estrogens examined, except for E3. The transforming activities of the estrogens, determined by the induced transformation frequencies, were ranked as follows: 4-OHE1 > 2-OHE1 > 4-OHE2 > 2-OHE2 > or = E2 or E1 > 2-MeOE1 or 16alpha-OHE1 > E3. Somatic mutations in SHE cells at the Na+/K+ATPase and /or hprt loci were induced only when the cells were treated with 4-OHE1, 2-MeOE1 or 4-OHE2 for 48 hr. Some estrogen metabolites induced chromosome aberrations in SHE cells following treatment for 24 hr. The rank order of the clastogenic activities of the estrogens that induced chromosome aberrations was 4-OHE1 > 2-OHE1 or 4-OHE2 > 2-OHE2 > E1. Significant increases in the percentage of aneuploid cells in the near diploid range were exhibited in SHE cells treated for 48 hr or 72 hr with each of the estrogens, except for 4-OHE1 and E3. Our results indicate that the transforming activities of all estrogens tested correspond to at least one of the genotoxic effects by each estrogen, i.e., chromosome aberrations, aneuploidy or gene mutations, suggesting the possible involvement of genotoxicity in the initiation of estrogen-induced carcinogenesis.

Aneuploidy↗
PubMed1996

Urinary markers of estrogen metabolism 2- and 16 alpha-hydroxylation in premenopausal women.

There is considerable scientific interest in whether measurement of the major estrogen metabolites 2- and 16 alpha-hydroxyestrone will shed light on the role of estrogen in the risk of breast cancer. These have been difficult to measure in large numbers because of the need for radiolabeled tracers, but a new assay is able to utilize spot urine samples. The main objective of this study was to assess the reliability of a newly developed enzyme immunoassay (EIA) for the measurement of 2- and 16 alpha-hydroxyestrone in urine samples collected from a large group of healthy premenopausal women enrolled in a clinical trial A secondary objective was to assess the impact of several factors such as body weight on the urinary estrogen metabolite ratios. The study cohort included 174 women aged 44-50, who were enrolled in the Cardiovascular Risk Factors and Menopause Trial, also referred to as the Women's Healthy Lifestyle Project (WHLP), an ongoing 5-year clinical trial of 535 premenopausal women randomized either to an intensive dietary life-style intervention group or to an assessment-only control group. Measurements of 2- and 16 alpha-hydroxyestrone showed a high intraclass correlation for blind duplicate urine samples (R = 0.94 and R = 0.80), cross-sectionally and over time (R = 0.79 and R = 0.62), in this population of healthy premenopausal women. The intervention diet (of 25% of total calories from fat) did not appear to influence the estrogen metabolite ratio. This new estrogen metabolite EIA demonstrates good reliability and thus may be appropriate for use in large epidemiologic studies of estrogen-related diseases. There was no relation between dietary fat reduction, weight loss, and increased exercise and change in the ratio among premenopausal women in this study.

Adult↗
PubMed1996

Superior and distinct antioxidant effects of selected estrogen metabolites on lipid peroxidation.

The effect of the estrogen metabolites, 4-hydroxyestrone and 17alpha-dihydroequilin (metabolites of estradiol-17beta and equilin, respectively), were examined for antioxidant effects on plasma and lipoprotein lipid peroxidation . Lipid peroxidation was evaluated by products of both fatty acid (thiobarbituric acid-reactive substances [TBARS]) and cholesterol (oxysterols) oxidation from lipoproteins or whole plasma. Although all estrogens significantly reduced lipid peroxidation, 4-hydroxyestrone was far more potent than either equilin or 17alpha-dihydroequilin in inhibiting TBARS formation in lipoproteins induced by Cu2+. Similar effects were also noted on TBARS formation in THP-l macrophages in culture. However, 17alpha-dihydroequilin (along with equilin) strongly inhibited oxysterol formation, whereas 4-hydroxyestrone was ineffective. These studies suggest that different estrogens might act preferentially on distinct lipid substrates in exhibiting antioxidant effects.

Antioxidants↗
PubMed1996

Bioactivation of estrone and its catechol metabolites to quinoid-glutathione conjugates in rat liver microsomes.

Although the carcinogenic effects of estrogens have been mainly attributed to hormonal properties, there is interest in estrogens acting as chemical carcinogens by binding to cellular macromolecules. In the present study, we explored factors which influence the rate of P450-catalyzed formation of the o-quinones (3,5-cyclohexadiene-1,2-diones) from 2-hydroxyestrone (2-OHE) and 4-hydroxyestrone (4-OHE) as well as from estrone in rat liver microsomes. The initially formed o-quinones were trapped as their GSH conjugates which were separated and characterized by HPLC with electrospray-MS detection. Two mono-GSH conjugates were observed from the 2-OHE-o-quinone as well as a conjugate where GSH had added twice to the molecule producing a di-GSH conjugate. 4-OHE-o-quinone gave only one mono-GSH adduct as well as a di-GSH adduct. Both 2-OHE and 4-OHE were excellent substrates for P450, generating o-quinone GSH adducts at 94 and 40 times, respectively, the rate of estrone. 2-OHE but not 4-OHE saturated P450 at unusually low concentrations (0.2 nmol of P450/mL) perhaps due to differences in the stability of the o-quinones formed in the active site of the enzyme. Preliminary data suggest that the o-quinones of both 2-OHE and 4-OHE could isomerize to quinone methides (4-alkyl-2,5-cyclohexadien-1-ones, QMs). The o-quinones of the catechol estrogens were incubated at 37 degrees C (pH 7.4) in the absence of GSH. Aliquots were removed at various times and combined with GSH. From the pseudo-first-order rate of disappearance of the o-quinone GSH adducts, the half-lives of the o-quinones were determined. The o-quinone from 2-OHE has a half-life of 42 +/- 3 s at 37 degrees C (pH 7.4), and the o-quinone from 4-OHE has a half-life of 12.2 +/- 0.4 min under identical conditions. The o-quinones of the AB ring analogs of the catechol estrogens (3,4-dihydroxy-5,6,7,8-tetrahydronaphthalene and 1,2-dihydroxy-5,6,7,8-tetrahydronaphthalene) isomerize to QMs, suggesting that a similar reaction pathway could occur with the o-quinones from catechol estrogens. In support of this, oxidation of 4-OHE and quenching with GSH after 70 min produced 9-dehydro-4-hydroxyestrone (3-hydroxy-1,3,5-(10),9(11)-estratetraen-17-one), a product which could result from either the QM hydrolysis product or the QM--glutathione conjugate, both of which could eliminate to give the conjugated alkene of 4-OHE. The implications of the o-quinone/QM pathway to the in vivo effects of catechol estrogens are not known; however, given the direct link between excessive exposure to endogenous estrogens and the enhanced risk of breast cancer, the potential for formation of additional reactive intermediates needs to be explored.

Animals↗
PubMed1999

Endocrine characteristics of human breast epithelial cells, MCF-10F.

MCF-10F is a spontaneously immortalized nontransformed human breast epithelial cell line which does not grow in soft agar or form tumors in nude mice. Though the presence of estrogen receptors has not been found in these cells, they can metabolize estradiol very efficiently. The present study describes the endocrine characteristics of this cell line with respect to growth response to estradiol and its metabolites, estradiol metabolism and aromatase activity. MCF-10F cells were growth stimulated by 16alpha-hydroxyestrone and estriol, whereas, estradiol and other estradiol metabolites did not affect cell proliferation. The constitutive level of 16alpha-hydroxyestrone, a metabolite of estradiol biotransformation that has been associated with enhanced carcinogenesis in several animal, cell and tissue culture models, was a hundredfold higher in the non-transformed MCF-10F cells than in the transformed MCF-7 cells. Treatment with the carcinogen, dimethylbenz(a)anthracene (DMBA), however, did not upregulate 16alpha-hydroxylation as was observed in transformed MCF-7 cells. MCF-10F cells also had no detectable aromatase activity though the level of 17-oxidation was unusually high as compared with MCF-7 cells. Our results using the non-transformed MCF-10F cells as a model system suggests that the presence of high level of 16alpha-hydroxyestrone, a metabolite previously shown to be associated with malignant phenotype, may not be sufficient for breast cancer transformation.

9,10-Dimethyl-1,2-benzanthracene↗