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PubMed2006

Urinary phytoestrogen excretion of rats bearing methylnitrosourea-induced mammary carcinoma in response to treatment with 2-methoxyestradiol.

The effect of treating mammary tumor-bearing rats with 2-methoxyestradiol (2-MeE2) on the urinary excretion of 12 phytoestrogens was investigated and compared with the changes in urinary excretion of estradiol metabolites. Alterations of excretion were registered for isoflavonoids, lignans and coumestans. However, due to large variations statistical significant differences were found only for two lignans, i.e. significant increases of enterodiol and matairesinol. Since the single components of phytoestrogens showed diverse alterations, excretions were expressed also by the ratio of total isoflavonoids to total lignans and compared with the estrogen ratios 2-hydroxyestrone to 16alpha-hydroxyestrone and A-ring to D-ring metabolites. The ratio of isoflavonoids to lignans was consistently decreased, whereas both ratios of estradiol metabolites were highly increased. The latter effect is probably due to demethylation of 2-methoxyestrone resulting in high catechol estrogen levels in urine. These results suggest that the high levels of catechol estrogens, produced by 2-MeE2 treatment, may have influenced the urinary excretion pattern of phytoestrogens.

2-Methoxyestradiol↗
PubMed2005

Comparison of plasma and urinary levels of 2-hydroxyestrogen and 16 alpha-hydroxyestrogen metabolites.

A modified ELISA assay for measurement of the two estrogen metabolites 2-hydroxyestrone (2OHE1) and 16alpha-hydroxyestrone (16alphaOHE1) in plasma and serum has been developed. Previously, these have only been measured in urine. It is not known how well the measurements of these metabolites in urine and plasma are correlated. The goal of this study was to compare urinary and plasma levels of 2OHE1 and 16alphaOHE1 and their ratios and to explore how they were affected by ethnicity, dietary and genetic factors, and medication use. Blood and urine samples were obtained from 511 nulliparous women, aged 17-35, from four ethnic groups during the same visit at the study center, on a random day of the menstrual cycle. The overall correlation between the 2OHE1/16alphaOHE1 ratio in plasma and urine was fair (rs = 0.52; p < 0.0001). In general, the correlation between the 2OHE1/16alphaOHE1 ratio in urine and plasma was higher among women not using oral contraceptives (OCs) (rs = 0.58; p < 0.0001) than among women currently using OCs (rs = 0.34; p < 0.0001). The correlation was highest for samples obtained during the mid-cycle in among non-OC users (rs = 0.83; p < 0.0001). Among non-OC users, the urinary 2OHE1/16alphaOHE1 ratio was stable over the menstrual cycle while there was an increase in the plasma 2OHE1/16alphaOHE1 ratio. The strongest factors predicting discordance between the urinary and plasma 2OHE1/16alphaOHE1 ratios among non-OC users were a baseline urinary 2OHE1/16alphaOHE1 ratio in the three upper quartiles (p < 0.001), the menstrual cycle phase (p = 0.001), and the number of cups of coffee consumed per day (p = 0.006). Among current OC users, the strongest predictors of discordance between the urinary and plasma 2OHE1/16alphaOHE1 ratios were a baseline urinary 2OHE1/16alphaOHE1 ratio in the three lower quartiles (p < 0.001), being black (p = 0.001), and being Asian (p = 0.014). In conclusion, we found that the correlation between the two methods was fair and varied according to the baseline urinary 2OHE1/16alphaOHE1 ratio, ethnic group, OC status, coffee consumption, and time of menstrual cycle when the samples were obtained.

Adipose Tissue↗
PubMed1998

Catechol estrogens as inhibitors of leukotriene synthesis.

Estrogens have a beneficial effect on atherosclerosis and osteoporosis after menopause, but their exact mechanism of action is still unknown. The aim of the present study was to investigate the effects of estradiol and its metabolites catechol estrogens on arachidonic acid metabolism in vitro. Estradiol had no effect on arachidonic acid metabolism up to 33 microM in A23187-stimulated human whole blood. All catechol estrogens (2-hydroxyestradiol, 2-hydroxyestrone, 4-hydroxyestradiol and 4-hydroxyestrone) had similar kinds of actions on arachidonic acid metabolism, being over ten times more potent inhibitors of leukotriene synthesis (IC50 values 0.044-0.16 microM) than thromboxane (IC50 values 0.99-2.1 microM) and prostaglandin E2 synthesis (IC50 values 0.84-5.5 microM). It is suggested that some of the protective actions of estrogens--e.g., on atherosclerosis and osteoporosis--may be related to the inhibition of leukotriene synthesis by catechol estrogens.

Arachidonate 5-Lipoxygenase↗
PubMed1999

Studies on neurosteroids. IX. Characterization of estrogens in rat brains using gas chromatography-tandem mass spectrometry.

The characterization of the classical estrogens (estrone, estradiol, estriol) and guaiacol estrogens (2-hydroxyestrone 3-methyl ether, 4-hydroxyestrone 3-methyl ether) in rat brains was performed using gas chromatography-tandem mass spectrometry (GC-MS-MS). Estrogens were purified from Wistar strain rat brains by some chromatographic pre-treatments, such as solid-phase extraction, preparative thin-layer chromatography or preparative high-performance liquid chromatography. After the derivatization with O-methylhydroxylamine and/or N,O-bis(trimethylsilyl)trifluoroacetamide, estrogens were identified by comparison of their chromatographic behavior during GC-MS-MS operating in the product ion scan mode and comparison with the product ion MS spectra of an authentic sample. These evidences suggested that estrogens exist in rat brains as neurosteroids or neuroactive steroids.

Animals↗
PubMed1997

Role of the estrogen receptor in the action of organochlorine pesticides on estrogen metabolism in human breast cancer cell lines.

As interest in the properties of xenoestrogenic compounds has grown, different in vitro cell culture systems have been proposed as models, against which to gauge relative estrogenic impact. Previous research indicated that some organochlorine-based pesticides elevated the production of 16 alpha-hydroxyestrone relative to 2-hydroxyestrone in ER+ MCF-7 breast cancer cells while phytochemicals like indole-3-carbinol reduced this ratio. That this ratio may be a biological marker of the risk of breast cancer has recently been demonstrated. In this study we have carried out the same paradigm in two ER- cell lines to examine the effect of receptor status. To determine whether the impact of chlorinated pesticides can be modulated by phytochemicals, the ability of indole-3-carbinol or brassinin to reverse the changes in metabolism was examined. Non-persisting phosphorus-based pesticides were also studied and shown not to have an effect on estrogen metabolism. The implications of these findings are examined.

Anticarcinogenic Agents↗
PubMed1999

Behavior and occurrence of estrogens in municipal sewage treatment plants--I. Investigations in Germany, Canada and Brazil.

The developed method enables the quantification of estrogens in sewage samples down to 1 ng/l and in river water down to 0.5 ng/l. Mean recoveries of the analytes in ground water after SPE extraction, clean-up and derivatization generally exceeded 75%. The determined R.S.D. varied from 0 to 14% at a spiking level of 0.05 microgram/l. Even in the raw influent and the final effluent from municipal STPs the mean recoveries of estrogens were mostly above 70%. Using this method the behavior and occurrence of natural estrogens and synthetic contraceptives in municipal sewage treatment plants (STP) were investigated in German and Canadian facilities. In the sewage of a German municipal STP close to Frankfurt/Main 17 beta-estradiol and estrone were determined, with mean concentrations of 0.015 microgram/l and 0.027 microgram/l, respectively. In two investigated municipal STPs, 17 beta-estradiol and 16 alpha-hydroxyestrone were eliminated with a higher efficiency than 17 alpha-ethinylestradiol and estrone. In Canadian and German STP discharges estrone, 17 beta-estradiol, 17 alpha-ethinylestradiol and 16 alpha-hydroxyestrone were frequently detected within the lower ng/l-range. A maximum concentration was found for estrone with 70 ng/l. In 15 investigated German rivers and streams only estrone was present with a maximum concentration of 1.6 ng/l.

Animals↗
PubMed2002

Impact of hormone replacement therapy on endogenous estradiol metabolism in postmenopausal women.

OBJECTIVES: Changes in estradiol metabolism may play a role in the pathophysiology of different diseases, of special interest being an increase in D-ring over A-ring metabolites for the risk of breast cancer. In the present work we investigated the effect of exogenous estradiol therapy on endogenous estradiol metabolism in postmenopausal women. METHODS: Three different studies were carried out in 126 women: in study A the women were treated for 4 weeks either with oral or with transdermal 17beta-estradiol, in study B for 4 weeks with oral or transdermal 17beta-estradiol sequentially combined with oral or transdermal norethisterone acetate, and in study C for 12 weeks either with oral 17beta-estradiol or with an oral continuous combination of 17beta-estradiol with the new progestin dienogest. As main representatives of the A- and D-ring metabolism, 2-hydroxyestrone (2-OHE1) and 16 alpha-hydroxyestrone (16-OHE1), respectively, were measured in 8 h night urine using enzyme immunoassay technique. RESULTS: Oral estradiol treatment resulted in a significant higher excretion of estradiol metabolites compared to transdermal treatment. Neither oral nor transdermal estradiol induced a significant change in the ratio of 16-OHE1 to 2-OHE1. The addition of oral or transdermal norethisterone acetate to estradiol did not alter on average the endogenous estradiol metabolism, although in individual patients a significant increase in 16-OHE1 metabolism was observed only with oral norethisterone. The continuous oral addition of dienogest did not lead to any significant change in estradiol metabolism. CONCLUSIONS: These results indicate that oral estradiol replacement therapy enhances the quantity of circulating estradiol metabolites. This may have a more negative impact on estrogenic target cells as compared to transdermal application. Progestin addition to estradiol replacement therapy seems to have no major impact on endogenous estradiol metabolism. Further studies, however, are necessary to evaluate the progestin effect in possible pre-disposed patients.

Estradiol↗
PubMed2004

Measuring seven endogenous ketolic estrogens simultaneously in human urine by high-performance liquid chromatography-mass spectrometry.

A rapid, sensitive, and specific high-performance liquid chromatography-electrospray ionization-multistage mass spectrometry (MS) method for measuring endogenous ketolic estrogen metabolites in human urine has been developed. The method requires a single hydrolysis/extraction/derivatization step and only 2.5 mL of urine, yet is able to simultaneously quantify estrone and its 2-methoxy and 2-, 4-, and 16alpha-hydroxy derivatives, 16-ketoestradiol, and 2-hydroxyestrone-3-methyl ether metabolites. The combination of a simple hydrazone derivatization step with multistage MS greatly enhances the sensitivity and specificity of the analysis of endogenous estrogen within human urine. Standard curves are linear over a 100-fold concentration range with linear regression correlation coefficients typically greater than 0.99. The lower limit of quantitation for each ketolic estrogen is 0.2 ng/2.5-mL urine sample (10 pg on column), with an accuracy of 93-103% and an overall precision, including the hydrolysis, extraction, and derivatization steps, of 1-13% relative standard derivation (RSD) for samples prepared concurrently and 8-16% RSD for samples prepared in separate batches. This method also allows for the identification of 2-hydroxyestrone-3-methyl ether in urine obtained from both pre- and postmenopausal women. This potentially protective estrogen metabolite has been previously reported only in the urine of pregnant women. Since individual patterns of estrogen metabolism may influence the risk of breast cancer, accurate and specific measurement of estrogen metabolites in biological matrixes will facilitate future research on breast cancer prevention, screening, and treatment.

Adult↗
PubMed1997

Estrogen-nucleic acid adducts: reaction of 3,4-estrone o-quinone with nucleic acid bases.

Metabolic activation of estradiol leading to the formation of catechol estrogens is believed to be a prerequisite for its genotoxic effects. Previous studies have shown that 3,4-estrone quinone (3,4-EQ) can redox-cycle and is capable of inducing exclusively single-strand DNA breaks in MCF-7 breast cancer cells [Nutter et al. (1991) J. Biol. Chem. 226, 16380-16386]. These studies, however, could not provide conclusive evidence about the mechanism of estrogen carcinogenesis. In order to explore this in more detail, we have shown previously that 3,4-EQ can react with adenine under electrochemical reductive conditions to yield an estrogen-nucleic acid adduct [Abul-Hajj et al. (1995) J. Am. Chem. Soc. 117, 6144-6145]. In this paper, we report the synthesis and identification of seven estrogen-nucleic acid adducts obtained from reaction of 3,4-EQ with adenine, thymine, and cytosine. Initial purification of reaction mixtures using TLC followed by HPLC gave sufficient quantities of reaction products for identification using 1H-NMR and mass spectral determinations. Reaction of 3,4-EQ with adenine, thymine, and cytosine gave the following estrogen-nucleic acid adducts: 8-(4-hydoxyestrone-1-yl)adenine, 3-adenylimino-1,5(10)-estradiene-4,17-dione,4-adenylimino-1, 5(10)-estradiene-3,17-dione, N1- [4-hydroxyestrone-1(alpha,beta)-yl]thymine, N4-(4-hydroxyestrone-1- yl)cytosine, and N4-(4-hydroxy- estrone-2-yl)cytosine. No reaction products were obtained with guanine presumably due to poor solubility in DMF.

Deoxyribonucleosides↗
PubMed1997

Mechanism of cytochrome P450-catalyzed aromatic hydroxylation of estrogens.

The mechanism of aromatic hydroxylation of estrogens by cytochrome P450 enzymes has been examined by comparing the oxidation of estrone with that of substrates carrying additional aromaticity such as equilenin and the structural analog 2-naphthol. Hamster liver microsomes preferentially catalyzed the conversion of estrone to 2-hydroxyestrone (Km = 30 and 25 microM and Vmax = 1497 and 900 pmol (mg of protein)-1 min-1 for 2- and 4-hydroxyestrone formation, respectively). In contrast, equilenin was hydroxylated exclusively at C-4 of the steroid ring system and 2-naphthol at the corresponding C-1 position (Km = 67 and 42 microM and Vmax = 2083 and 3226 pmol (mg of protein)-1 min-1 for 4-hydroxyequilenin and 1,2-dihydroxynaphthalene formation, respectively). This shift in the specificity of hydroxylation was due to the introduction of additional aromaticity at ring B of equilenin, because hamster liver microsomes are known not to contain any estrogen-4-hydroxylase, only estrogen-2-hydroxylase activity catalyzed by cytochrome P450 3A family enzymes. The exclusive 4-hydroxylation of equilenin is proposed to be due to a preferred delocalization of the naphthoxy radical an intermediate in the hydroxylation, to C-4, whereas delocalization to C-2 requires additional activation energy and is energetically not favored. Based on these electronic considerations, a mechanism of aromatic hydroxylation of estrogens is proposed which features hydrogen abstraction from the phenolic hydroxy group, electron delocalization of the phenoxy radical to a carbon-centered radical, and subsequent formation of catechol metabolites by hydroxy radical addition at C-2 or C-4 depending on steric or electronic constraints.

Animals↗
PubMed1998

Inhibition of glutathione S-transferase activity by the quinoid metabolites of equine estrogens.

The risk factors for women developing breast and endometrium cancers are all associated with a lifetime of estrogen exposure. Estrogen replacement therapy (ERT) in particular has been correlated with a slight increased cancer risk, although the numerous benefits of ERT may negate this harmful side effect. Equilenin and equilin are equine estrogens which make up between 30% and 45% of the most widely prescribed estrogen replacement formulation, Premarin (Wyeth-Ayerst). In this study we have synthesized the catechol metabolites of equilenin [4-hydroxyequilenin (4-OHEN)] and equilin [4-hydroxyequilin (4-OHEQ)] and examined how changing unsaturation in the B ring affects the formation of o-quinone GSH conjugates and the ability of the o-quinones and/or GSH conjugates to inhibit glutathione S-transferase (GST). Interestingly, both 4-OHEN and 4-OHEQ autoxidized to o-quinones without the need of oxidative enzyme catalysis. 4-OHEN-o-quinone reacts with GSH to give two mono-GSH conjugates and one diadduct. The behavior of 4-OHEQ was found to be more complex than 4-OHEN as conjugates resulting from 4-OHEN were detected in addition to the 4-OHEQ GSH adducts. Both 4-OHEN and 4-OHEQ were found to be potent inhibitors of GST-catalyzed conjugation of GSH with 1-chloro-2,4-dinitrobenzene. In contrast, the endogenous catechol estrogens, 4-hydroxyestrone (4-OHE) and 2-hydroxyestrone (2-OHE), were without effect unless tyrosinase was present to convert the catechols to o-quinones. Scavengers of reactive oxygen species and metal chelators had no effect on GST inhibition by catechol estrogens with the exception of the catalase which protected GST activity. Kinetic studies showed that 4-OHEN was a potent irreversible inactivator of GST. Preincubation of the enzyme with 4-OHEN showed a time-dependent increase in inhibitory effect, and gel filtration did not restore GST activity confirming the irreversible nature of the enzyme inactivation. Analysis of the Kitz-Wilson plot gave a dissociation constant of the reversible enzyme-inhibitor complex (Ki = 620 microM) and a rate constant of conversion of the reversible enzyme-inhibitor complex to the irreversibly inhibited enzyme (k2 = 7.3 x 10(-)3 s-1). These data suggest that 4-OHEN is an irreversible inactivator with relatively low affinity for GST; however, once formed the 4-OHEN enzyme complex is rapidly converted to the irreversibly inhibited enzyme. The inhibition mechanism likely involves oxidation of the catechol estrogens to o-quinones and covalent modification and/or oxidation of critical amino acid residues on GST. In addition, hydrogen peroxide generated through redox cycling of the o-quinone and/or semiquinone radical and GSH could cause oxidative damage to GST.

Animals↗
PubMed2004

Physical activity, body size, and estrogen metabolism in women.

OBJECTIVES: Physical activity has demonstrable effects on estrogen levels in pre- and postmenopausal women. Increased oxidation of estrone to 2-hydroxyestrone (2HE) relative to 16alpha-hydroxyestrone (16HE) has been hypothesized to reduce breast cancer risk, but little is known about the effect of physical activity and body size in relation to the ratio of 2HE and 16HE in women. We examined these relationships in cross-sectional analyses of 157 North American and Chinese women. METHODS: Physical activity was assessed using validated questionnaires. Adiposity was assessed as body mass index (BMI, kg/m2) and by anthropometric methods (% body fat). Estrone metabolites, 2HE and 16HE, were determined from urine via ELISA. RESULTS: Regression analyses on the 2HE/16HE ratio revealed an interaction between leisure-time physical activities and adiposity in both North American and Chinese women (p < or = 0.05). Women reporting low levels of leisure-time physical activity who had higher BMI levels had 2HE/16HE ratios that were lower than their lean counterparts. In contrast, women with higher BMI levels that were physically active maintained higher 2HE/16HE ratios. CONCLUSION: These findings suggest that physical activity participation has the potential to modify the adverse effect of increased adiposity on estrogen metabolism in North American and Chinese women.

Adipose Tissue↗
PubMed1999

[The significance of estrogen metabolism for hormone-dependent neoplasms from the clinical-pharmacological viewpoint].

The present review summarizes existing data regarding estrogen metabolism and hormone-responsive cancers. Endogenous estradiol breakdown takes place by oxidation at the A-ring and the D-ring; the resulting metabolites, however, are not inactive substances, as assumed formerly, but elicit different biological effects, with A- and D-ring metabolites to some extent developing opposite actions. Multiple results are available showing stimulating and inhibiting properties of some metabolites on the growth of cancer. Among the D-ring metabolites are substances with proliferation-stimulating properties, e.g. 16 alpha-hydroxyestrone, and among the A-ring metabolites, the catechol estrogens, compounds with inhibiting properties. There are indications that endogenous production of proliferation-stimulating metabolites, especially 16 alpha-hydroxyestrone, which has been shown to be mutagenic, is increased in some cancers. In these cases the ratio of A- to D-ring metabolism is shifted in favour of the D-ring metabolites. This ratio can be influenced by various substances e.g. by indole-3-carbinol, an ingredient of cruciferous vegetables, which can increase the body's own production of catechol estrogens. However, as yet only a few metabolites have been investigated with respect to their influence on cancer development and growth. Presumably other metabolites may also be able to interfere directly or indirectly with cancer progression. Thus a great deficit in research work remains in this field. The existing findings on the pathophysiology of estradiol metabolism on the development and progression of cancer offer promising opportunities for clinical-pharmacological intervention in the treatment of human cancer. 2-Methoxyestradiol, physiologically present at low concentrations, seems to be of special interest. Several investigations were able to demonstrate that 2-methoxyestradiol inhibits proliferating tumoric tissues. Inhibition of neoangiogenesis and inhibition of tubulin polymerization have been revealed as mechanisms. Specific studies on the clinical use of this estradiol metabolite seem to be justified.

Animals↗
PubMed2002

Stimulatory effect of cigarette smoking on the 15 alpha-hydroxylation of estradiol by human term placenta.

OBJECTIVE: Our objective was to characterize the oxidative metabolism of estradiol by human term placenta and its modulation by cigarette smoking. METHODS: Placental microsomes were prepared from term placentas obtained from 13 cigarette smokers (20 to 30 cigarettes per day until the time of delivery) and 13 control subjects who were nonsmokers. Estrogen metabolism was studied by incubation of 250 nmol/L [(3)H]estradiol with placental microsomes and NADPH, and the estrogen metabolites were determined by HPLC and gas chromatography-mass spectrometry. RESULTS: 2-Hydroxyestradiol was the major hydroxyestrogen detected, followed by 6alpha-hydroxyestradiol. Small amounts of several other hydroxyestrogen metabolites (4-hydroxyestradiol, 6beta-hydroxyestradiol, 7alpha-hydroxyestradiol, and 16alpha-hydroxyestradiol) were also detected. Large amounts of estrone plus small amounts of 2-hydroxyestrone and unidentified nonpolar metabolites were formed. Cigarette smoking stimulated the placental hydroxylation of benzo[a ]pyrene by about 16-fold. Cigarette smoking had little or no effect on the overall rate of placental estradiol metabolism or on the formation of estrone, 2-hydroxyestradiol, 2-hydroxyestrone, or 16alpha-hydroxyestradiol. However, placental formation of 4-hydroxyestradiol and 7alpha-hydroxyestradiol was increased 38% (P =.08) and 150% (P =.05), respectively, in cigarette smokers. The formation of 6alpha-hydroxyestradiol was decreased 33% (P =.04). Metabolic formation of 15alpha-hydroxyestradiol was observed during incubations of estradiol with placental microsomes from 11 of the 13 cigarette smokers, but this metabolite was not detected during incubations with placental microsomes from any of the 13 nonsmokers. Analysis of data from all 26 placentas showed that the 15alpha-hydroxylation of estradiol was highly correlated with benzo[a ]pyrene hydroxylation (r = 0.93; P <.001). CONCLUSIONS: Many hydroxylated estradiol metabolites were formed by placental microsomes from cigarette smokers and nonsmokers. 15alpha-Hydroxylation of estradiol was markedly stimulated in the placentas of cigarette smokers.

Adult↗
PubMed1997

Expression and functional domains of rabbit liver UDP-glucuronosyltransferase 2B16 and 2B13.

Southern blot analysis has demonstrated that the 5' portion of the rabbit liver dexamethasone-inducible UDP-glucuronosyltransferase (UGT) 2B13 RNA is related in sequence to a family of UGT genes (Tukey, R. H., Pendurthi, U. R., Nguyen, N. T., Green, M. D., and Tephly, T. R. (1993) J. Biol. Chem. 268, 15260-15266). To identify these additional gene transcripts, rabbit liver cDNA libraries were screened with a 5' conserved 330-base pair UGT2B13 cDNA fragment, resulting in the isolation and characterization of several rabbit liver UGT cDNAs. One such clone, called pGT11, encodes a putative glycoprotein that is 78% similar to rabbit UGT2B13. The new UGT has been designated UGT2B16. The UGT2B16 gene is expressed as a single 4200-base RNA transcript that is regulated only in adult rabbits. The predicted NH2-terminal 25 amino acids of UGT2B16 are identical to that of rabbit liver UGT2B13, with the remainder of the protein being 77% similar to UGT2B13. Expressed UGT2B16 protein in COS-1 cells was active toward 4-hydroxybiphenyl, similar to that of UGT2B13. However, UGT2B16 efficiently conjugated 4-hydroxyestrone and 4-tert-butylphenol, substrates that are not efficiently catalyzed by UGT2B13. To further characterize the structural domains of UGT2B16 and UGT2B13, a series of chimeric cDNAs were constructed that contained portions of both UGT2B16 and UGT2B13. Chimeric 2B163002B13531, which contained the amino-terminal UGT2B16 amino acids 1-300 followed by amino acids 301-531 of UGT2B13, as well as chimeric 2B163582B13531 and 2B164342B13531 proteins, catalyzed the glucuronidation of 4-hydroxyestrone, indicating that the carboxyl terminus of UGT2B13 could substitute for those same regions on UGT2B16. However, the replacement of the carboxyl end of UGT2B13 with 2B16300-531 or 2B16434-531 dramatically impaired the catalytic function of the chimeric proteins. These results indicate that the carboxyl end of UGT2B13 plays an important role in the functional and possible conformational state of the protein.

Amino Acid Sequence↗
PubMed2001

Quantitative determination of 3,3'-diindolylmethane in urine of individuals receiving indole-3-carbinol.

Indole-3-carbinol (I3C) or, more correctly, its acid condensation products act as chemoprotective agents via several mechanisms. It induces the expression of cytochrome P-450 1A1, which shifts the estrogen metabolic pathway in favor of C-2 hydroxylation and away from the formation of 16 alpha-hydroxyestrone, a suspected endogenous carcinogen. Increased 16 alpha-hydroxylation of estrogen is associated with greater risk of cancer of the cervix, breast, endometrium, and larynx. The production of 4-hydroxyestrone is also inhibited by I3C. I3C can induce a G1 cell cycle arrest in human MCF-7 breast cancer cells. It can suppress aberrant crypt foci. I3C significantly inhibits the cell adhesion, spreading, and invasion associated with an upregulation of PTEN (a tumor suppressor gene) and E-cadherin (a regulator of cell-cell adhesion) expression in T47-D human breast cancer cells. Thus I3C exhibits anticancer activities by suppressing breast tumor cell growth and metastatic spread. A gas chromatography-mass spectrometry method for the quantitation of diindolylmethane, the principal acid condensation product of I3C, has been developed for use in determining compliance in subjects who have been treated with I3C. The method utilizes a 1-ml urine sample. We have used this method to correlate I3C ingestion with regression of cervical intraepithelial neoplasia in a population of women at risk for cervical cancer. The assay provides an objective marker of consumption using a noninvasive biological fluid and illustrates that diindolylmethane may be used as a marker of compliance in I3C dietary intervention studies.

Anticarcinogenic Agents↗
PubMed2004

High levels of oxidative DNA damage in lymphocyte DNA of premenopausal breast cancer patients from Egypt.

Egypt shows a parallel increase in premenopausal breast cancer and environmental pollution. The purpose of this study is to explore a possible relationship between oxidative DNA damage, urinary estrogen metabolites and breast cancer in Egyptian premenopausal women. We conducted a pilot study of Egyptian breast cancer involving 29 cases and 32 controls and analysed lymphocyte DNA levels of 7,8-dihydro-8-oxo-2'-deoxyguanine (8-oxo-dG), a measure of oxidative DNA damage using high performance liquid chromatography with electro-chemical detection (HPLC-ECD) method. We analysed levels of urinary estrogen metabolites, 2-hydroxyestrone (2-OHE) and 16alpha-hydroxyestrone (16alpha-OHE) by an enzyme immuno assay. We also collected residential, occupational, and reproductive histories of all study subjects. We detected, in all subjects, exceptionally high levels of 8-oxo-dG and thus oxidative DNA damage, the levels (mean 8-oxo-dG/10(5) dG+/-SD) were significantly (P<0.01) higher in breast cancer cases (139.4+/-78.4) than in controls (60.9+/-51.5). Urinary 2-OHE and 16alpha-OHE or their ratio was not significantly different between cases and controls. However, 8-oxo-dG levels were positively correlated (P<0.05) with 2-OHE and 16alpha-OHE from cases while controls showed a negative correlation (P<0.05). Urban residence (Odds Ratio [OR] 3.1; Confidence interval [CI], 1.1-9.3), infertility (OR [9.8]; CI [1.1-89.7]), age (OR [2.6]; CI [1.4-4.6]) and 8-oxo-dG (OR 5.8; CI 1.9-17.5) levels were found to be significant predictors of breast cancer. Our finding of exceptionally high levels of 8-oxo-dG, a common result of oxidative DNA damage, warrant future studies on a larger population of premenopausal women in Egypt with consideration of other susceptibility markers and dietary characteristics.

Adult↗
PubMed2006

Soy isoflavonoid effects on endogenous estrogen metabolism in postmenopausal female monkeys.

Endogenous estrogens are important determinants of breast cancer risk in postmenopausal women. In this study we evaluated the effects of dietary soy isoflavonoids on endogenous estrogen metabolism in a postmenopausal primate model. Ovariectomized female cynomolgus monkeys were randomized to receive one of three diets for 36 months: (i) isoflavonoid-depleted soy protein isolate (SPI-) (n = 29); (ii) soy protein isolate with 129 mg isoflavonoids/1800 kcal diet (8.6 mg isoflavonoids/kg body weight (BW), expressed in aglycone units) (SPI+) (n = 29) or (iii) isoflavonoid-depleted soy protein isolate with conjugated equine estrogens (CEE) at a dose of 0.625 mg/1800 kcal diet (0.042 mg CEE/kg BW) (n = 30). Mean plasma isoflavonoid concentrations in the SPI+ group were 946.9 +/- 135.9 nmol/l, and equol was the primary circulating isoflavonoid (549.7 +/- 61.6 nmol/l). The SPI+ diet resulted in lower serum estrone (E(1)) after 29 (-26%, P = 0.03) and 34 months (-21%, P = 0.04) compared to the SPI- diet, while urinary 2-hydroxyestrone (P = 0.005) and the 2 to 16alpha-hydroxyestrone ratio (P < 0.0001) were markedly higher in the SPI+ group compared to SPI-. Isoflavonoid treatment did not significantly alter gene markers of estrogen metabolism or estrogen receptor agonist activity in breast tissue. Within the SPI+ group, higher concentrations of serum equol (but not daidzein or genistein) corresponded to significantly lower serum E(1), mammary gland epithelial area and uterine weight (P < 0.01 for all). These findings suggest that long-term exposure to soy isoflavonoids, equol in particular, may facilitate endogenous estrogen clearance and catabolism to more benign 2-hydroxylated metabolites.

Animals↗