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PubMed1983

The effect of hypocholesterolemic drug treatment on adrenocortical cell function.

The role of exogenous lipoprotein cholesterol versus endogenous cholesteryl esters as substrates in adrenal steroidogenesis was studied in isolated rat adrenal cells. Hypocholesterolemic drugs were used in rats to depress the plasma cholesterol concentration and the adrenal cholesterol concentration. Adrenal cortical cells were prepared in the usual way. The steroidogenic response to ACTH in normal adrenal cells and in cells which have been cholesterol-depleted was studied. Normal adrenal cells responded specifically over a 6 h incubation period to low doses of ACTH (half-maximal response equivalent to 40 microunits ACTH). These normal cells exhibited no altered response over a 3 h period to ACTH in the presence of serum or serum lipoproteins. The hypocholesterolemic drugs, 4-aminopyrazolo-[3,4-d]-pyrimidine, hexestrol and 17 alpha-ethinyl estradiol were used to lower plasma cholesterol, and after 1 day of 4-aminopyrazolo-[3,4-d]-pyrimidine and 5 days of hexestrol or 17 alpha-ethinyl estradiol treatment the plasma total cholesterol concentrations were similar. After 3 days of 4-aminopyrazolo-[3,4-d]-pyrimidine treatment the adrenal total cholesterol content was lower than after 1 day of this treatment, or 5 days of hexestrol treatment or 5 days of 17 alpha-ethinyl extradiol treatment. Lipoproteins had no significant effect on ACTH-stimulated steroidogenesis in cells isolated from rats treated for 1 day with 4-aminopyrazolo-[3,4-d]-pyrimidine, or for 5 days with hexestrol or 17 alpha-ethinyl estradiol. However, lipoproteins did stimulate steroidogenesis in cells from rats treated for 3 days with 4-aminopyrazolo-[3,4-d]-pyrimidine. The results show that normal adrenal cells contain a reserve of intracellular cholesterol so that the supply of endogenous cholesterol for steroidogenesis does not limit the response to ACTH and exogenous lipoproteins have no effect on steroidogenesis. However, if the cells are severely depleted of cholesterol then exogenous lipoproteins must be added for maximal steroidogenesis to occur.

Adrenal Cortex↗
PubMed1985

Synthesis and receptor-binding affinity of fluorotamoxifen, a possible estrogen-receptor imaging agent.

Aminotamoxifen was totally synthesized from p-nitrobenzoyl chloride via a Friedel-Crafts acylation. Then, by means of a Balz-Schiemann reaction, aminotamoxifen was converted into fluorotamoxifen. The triazene variation of this conversion, with a 25% yield, enables a rapid, one-step diazotization, incorporating a fluorine atom into the phenyl ring of the tamoxifen. This reaction may be useful for the preparation of low specific activity 18F-labeled tamoxifen, for distribution, and for estrogen-receptor studies. For these in vivo and in vitro studies, fluorotamoxifen was also synthesized from p-fluorobenzoyl chloride, and its chemical intermediates were compared with estradiol and hexestrol, for their receptor binding and competition, as well as for their uterotropic activity. It is demonstrated that tamoxifen and fluorotamoxifen are strong estradiol agonists and partial hexestrol agonists, while aminotamoxifen is a weak estradiol and hexestrol agonist.

Animals↗
PubMed1980

Receptor-binding radiopharmaceuticals for imaging breast tumors: estrogen-receptor interactions and selectivity of tissue uptake of halogenated estrogen analogs.

Four halogenated estrogen analogs--o-fluorohexestrol, and 1-fluoro-, 1-bromo-, and 1-iodohexestrol--have been prepared and tritium-labeled in high specific activity, to investigate their potential as estrogen-receptor-based agents for imaging breast tumors. These compounds bind with high affinity in vitro to the cytoplasmic uterine estrogen receptor from rat and lamb and sediment as 8S receptor complexes on sucrose gradients. After 1 hr in immature rats, these compounds show high uptake into the uterus, but low uptakes (10--25% of the uterine levels) into most nontarget tissues. The uterine uptake is estrogen specific since it is depressed by excess nonradioactive estradiol. Uptake selectivity is greatest for the fluorohexestrols and decreases for the bromo and iodo compounds. In mature rats bearing DMBA-induced mammary tumors, selective uptake by the uterus and tumors is seen with 1-fluoro[3H4]hexestrol and o-fluoro[3H3]hexestrol. The studies indicate that these four halogenated hexestrols are promising candidates as estrogen-receptor-based agents for the imaging of human breast tumors.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1997

Different residues of the human estrogen receptor are involved in the recognition of structurally diverse estrogens and antiestrogens.

We have previously examined, by alanine scanning mutagenesis, amino acids 515-535 of the estrogen receptor (ER) ligand binding domain to determine which of these residues are important in estradiol binding. Mutation at four sites that potentially lie along one face of an alpha-helix, Gly521, His524, Leu525, and Met528, all significantly impaired estradiol binding by the ER (Ekena, K., Weis, K. E., Katzenellenbogen, J. A., and Katzenellenbogen, B. S. (1996) J. Biol. Chem. 271, 20053-20059). In this report, we compare the pattern of residues that are important in the recognition of several structurally diverse estrogen agonists and antagonists (the synthetic nonsteroidal agonist hexestrol, an agonist derived from the mold metabolite zearalenone, P1496, and the partial agonist-antagonist trans-hydroxytamoxifen) with those that are predicted to contact estradiol in the receptor-ligand complex. Although there are some similarities in the pattern of residue recognition among all four ligands, each ligand showed distinct differences as well. Interestingly, alanine substitution at only one residue, the leucine at position 525, was found to inhibit binding of all the ligands tested. Another residue, His524, was found to be important in the recognition of three different agonists but not trans-hydroxytamoxifen (the only ligand lacking a second hydroxyl group). The recognition of estradiol and another agonist, P1496, was impaired by the G521A mutation, whereas ligand-induced activity by the two compounds that lack B- and C-rings, hexestrol and trans-hydroxytamoxifen, was unaffected. Our findings demonstrate that these ligands fit into the ER ligand binding pocket differently and that each contacts a distinct set of amino acids. The smaller ligands (estradiol and hexestrol) have a narrower footprint of interacting residues than the larger ligands (P1496 and trans-hydroxytamoxifen). This pattern of interaction is most consistent with the amino acids within this region being in contact with the portion of these ligands that corresponds to the D-ring end of estradiol. The interplay between the shape of an ER ligand and the residues that support its binding to ER may potentially underlie the selective actions of different ER ligands in various cell and promoter contexts.

Binding Sites↗
PubMed1991

Qualitative study of the interaction mechanism of estrogenic drugs with tubulin.

Synthetic estrogenic drugs (E-diethylstilbestrol, erythro-hexestrol and E,E-dienestrol) inhibit tubulin assembly and erythro-hexestrol and E,E-dienestrol lead to the formation of twisted ribbon structures. For the inhibitory effect on tubulin assembly, estrogenic drugs seem to interact directly with tubulin 6S on site(s) analogous to the colchicine-site, but independent of the GTP- and vinblastine-sites. This binding does not involve tubulin tryptophanyl residues or sulfhydryl groups. The influence of temperature, calcium and magnesium on the formation of twisted ribbon structures induced by the binding of estrogenic drugs to microtubular protein and tubulin has also been studied. This formation is strongly magnesium-dependent whereas preformed twisted ribbon structures are calcium- and chilling-insensitive.

Binding Sites↗
PubMed1980

Adrenal estrogens in relation to ovarian function.

The effect of gonadotropins and ovarian hormones on adrenal estrogens in female rats was examined in relation to the ovarian function. Pregnant mare serum gonadotropin (PMS) increased adrenal estradiol level in intact rats but did not alter estrone levels. Human chorionic gonadotropin (HCG) with PMS did not alter the estrone and estradiol levels. Neither PMS or HCG with PMS affected these steroid levels in ovariectomized rats. Injection of hexestrol to intact rats decreased the estrone level. Further decrease in the estrone level resulted after administration of hexestrol with progesterone. In a developmental study, the estrone level at 35 days of age was higher than that at 50 and 80 days of age. The estrone level gradually decreased with adrenal development after ovariectomy performed at 21 days of age. When adrenal slices were incubated with either 3H-estrone or 3H-estradiol, higher radioactivity of estrone accumulated in the adrenal gland of immature rats than in that of adult rats. There was a larger conversion of 3H-estradiol to 3H-estrone than the reverse process in both immature and adult rats. These results indicate that the adrenal estrogens were influenced by ovarian function.

Adrenal Glands↗
PubMed1978

Lysosomal phospholipids from rat liver after treatment with different drugs.

Rats were treated with 5 different drugs p-ethoxyacetanilide (I), indometacin (II) and nor-amidopyrine-methanesulfonate (III), O,O'-bis(diethylaminoethyl)hexestrol(IV) and choloroquine (V) for 3 - 4 weeks. Liver cell fractions were isolated by discontinuous gradient centrifugation and the specific activity of acid phosphatase was determined in each. Lysosomal fractions contained widely varying amounts of this marker enzyme, indicating that the concentration of lysosomes within these fractions differed. The amounts and patterns of phospholipids reflected this fact. Since we assumed bis(monoacylglycero)phosphate [(MAG)2-P; synonym:lysobisphosphatidic acid] is a marker lipid for secondary lysosomes, we expected and found significant quantities of this acidic phospholipid only in those lysosomal fractions which were also rich in acid phosphatase activity. 12% of the lysosomal phospholipids from animals receiving the hexestrol derivative (IV), and 19% of those from the chloroquine (V) experiment were present as (MAG)2P. The fatty acid compositions of this lysosomal phospholipid were not the same in all lysosome fractions. The more (MAG)2P present in the lysosomes, the more unsaturated are the fatty acids. Thus, after treatment with chloroquine, more than 90% of the fatty acids from (MAG)2P are unsaturated; C22:6 represents about 70% of the total.

Acid Phosphatase↗
PubMed1985

Amphiphilic cationic drugs and phospholipids influence the activities of beta-galactosidase and beta-glucosidase from liver lysosomal fraction of untreated rats.

The influence of amphiphilic drugs and phospholipids on the activities of beta-galactosidase and beta-glucosidase from liver lysosomal fractions of untreated rats, isolated by affinity chromatography using castor bean lectins, was studied in vitro. Chloroquine (93 microM) inhibited beta-galactosidase activity by about 30%, while O,O'-bis(diethylaminoethyl)hexestrol showed no inhibitory effect. Neutral phospholipids (phosphatidylcholine, phosphatidylethanolamine, sphingomyelin) inhibited the enzyme slightly, while the enzyme activity was drastically reduced in the presence of acidic phospholipids (phosphatidylinositol, phosphatidylserine, bis-(monoacylglycero)phosphate). Lysosomal beta-glucosidase was strongly inhibited by chloroquine and O,O'-bis(diethylaminoethyl)-hexestrol. The neutral phospholipids showed only a moderate inhibitory effect, whereas the acidic phospholipids were stimulators. Bis(monoacylglycero)phosphate was by far the best stimulating compound.

Animals↗
PubMed1990

Uterine adenocarcinoma in mice following developmental treatment with estrogens: a model for hormonal carcinogenesis.

In order to study the effects of perinatal exposure to estrogens on the developing reproductive tract, outbred female mice were treated neonatally (days 1 to 5) with varying doses of diethylstilbestrol (DES) and sacrificed from 1 to 18 months of age. Uterine adenocarcinoma was observed in a time- and dose-related manner after DES treatment; at 18 months, neoplastic lesions were seen in 90% of the mice exposed neonatally to 2 micrograms/pup of DES/day, while none was observed in the corresponding control mice. These DES-induced uterine tumors were estrogen dependent; when DES-treated mice were ovariectomized before puberty, no uterine tumors developed. As a marker for neoplasia, uterine tumors were transplanted and carried as serial transplants in nude mice. The transplanted tissue retained some differentiated uterine gland structure and function and also required estrogen supplementation for maintenance. Additional groups of neonatal mice were treated with various DES analogues (hexestrol and tetrafluorodiethylstilbestrol) and steroidal estrogens. The compounds were ranked according to developmental estrogenic potency (hexestrol greater than trifluorodiethylstilbestrol greater than DES greater than 17 beta-estradiol). The combined prevalence of uterine atypical hyperplasia and adenocarcinoma follows the order of estrogenic potency. The experimental induction of these tumors will provide the basis for additional studies in mechanisms of hormonal carcinogenesis.

Adenocarcinoma↗
PubMedDate not supplied

The use of liquid membranes in the multi-residue extraction of stilbenes in a variety of biological matrices and their detection with LC-ES-MS.

Supported liquid membrane (SLM) has been used as a sample preparation method in the simultaneous extraction of a mixture of three stilbene compounds in cow's milk, urine, bovine kidney and liver tissues matrices. The stilbene compounds analysed included, dienestrol, diethylstilbestrol and hexestrol. The liquid membrane used for trapping these compounds consisted of 5% tri-n-octylphosphine oxide (TOPO) dissolved in di-n-hexylether/n-undecane (1:1). The extraction efficiencies obtained after enrichment of 1 ng/L stilbenes in variety of biological matrices of milk, urine, liver, kidney and water, ranged from 60 - 70%, 71 - 86%, 69 - 80%, 63 - 7A% and 72 - 93% respectively. The detection limits obtained from urine extraction were 2.1 ng/L, 1.3 ng/L and 3.0 ng/L; from liver and kidney tissues were 2.9 ng/L, 1.6 ng/L and 3.8 ng/L and from milk was 3.2 ng/L, 2.5 ng/L and 4.3 ng/L for hexestrol, dienestrol and diethylstilbestrol respectively.

Animals↗
PubMed1982

Deuterium labeling of diethylstilbestrol and analogues.

E-2,2,3',3'',5,5,5',5''-octadeuteriodiethylstilbestrol (DES-d8) and Z-2,3'3'',4,5,5,5',5''-octadeuterio-3,4-bis(p-hydroxyphenyl)-2-hexene (psi-DES-d8) were synthesized from E-diethylstilbestrol (DES) by hydrogen/deuterium exchange in a mixture of methanol-d and deuterium chloride in deuterium oxide. The structures, isotopic purity, and positions of uptake of deuterium were determined by nuclear magnetic resonance (NMR) and mass spectrometry (MS). Additional confirmation of the positions of deuterium exchange in stilbestrols was obtained from an analysis of the oxidation of dES-d8 to Z,Z-2,3',3'',5,5',5''-hexadeuteriodienestrol (beta-DIES-d6) and of the hydrogen/deuterium exchange reaction of hexestrol (HEX) to 3',3'',5',5''-hexestrol, (HEX-d4). Structural analysis and the determination of isotopic purity of the latter two compounds were also carried out by NMR and MS. The uptake of eight deuterium atoms by DES is postulated to proceed via two different reactions occurring simultaneously: 1. acid catalyzed deuteration of all four phenolic ortho-positions (3',3'',5',5''); 2. acid catalyzed deuteration of the olefin bridge with subsequent formation of deuterated psi-DES (3 or 4). Due to the equilibration between DES, psi-DES, and Z-diethylstilbestrol (cis-DES) in the acidic reaction mixture at 85 degrees C, the deuterated psi-DES is thought to rapidly rearrange to deuterated DES. Repeated deuteration will eventually form DES-d8 fully labeled in the 2,2,5,5 methylene positions.

Deuterium↗
PubMed1986

Mitotic inhibition and aneuploidy induction by naturally occurring and synthetic estrogens in Chinese hamster cells in vitro.

We used a predominantly diploid Chinese hamster cell line to test a number of naturally occurring and synthetic estrogens for their ability to arrest cells at metaphase, their potential for allowing anaphase recovery, and their capability of inducing aneuploid progeny. The chemicals employed included diethylstilbestrol, dienestrol, hexestrol, beta-estradiol, ethynylestradiol and estriol. We also tested progesterone, estrone and testosterone in this regard. Only estrogens and their synthetic analogs caused mitotic arrest and aneuploidy, while progesterone, estrone and testosterone did not cause mitotic disturbances. Among the estrogens, DES was the most effective arrestant on a comparative molar basis, whereas dienestrol was most potent over a wide range of concentrations. Estriol was the least potent as an arrestant but was an effective inducer of aneuploidy. The addition of a metabolic activator (S9) did not alter the ability of DES to arrest mitosis. Following the removal of the drugs, cells were able to quickly reorganize a spindle apparatus and enter anaphase. Diethylstilbestrol, dienestrol, hexestrol, beta-estradiol, ethynylestradiol and estriol caused significant increase in aneuploidy within a narrow range of high concentrations in recovering cell populations. Aneuploidy was induced in a non-random manner. Immunofluorescence studies with anti-tubulin antibody indicate that estrogens may have a mechanism of mitotic arrest similar to that of colchicine and colcemid, viz inhibiting the polymerization of tubulin to form microtubules. These data suggest that the interaction between estrogens and microtubules may mediate the induction of aneuploidy in somatic cells. Aneuploidy induction by DES and similar compounds may be related to their carcinogenic potential.

Anaphase↗
PubMed2003

Pharmacological inhibition of outwardly rectifying Cl- currents in rat peritoneal mast cells: a comparison of different stilbene derivatives.

Diethylstilbestrol and other stilbene derivatives can provide some inhibition of the outwardly rectifying Cl- current (I(Cl-,OR)) in rat peritoneal mast cells. In order to elucidate structure-activity relationships of diethylstilbestrol, 12 stilbenes as well as 17beta-estradiol and hexestrol were tested in rat peritoneal mast cells using the nystatin-perforated patch approach of the whole-cell patch-clamp technique. Since trans-stilbene showed no effect, the substituents of diethylstilbestrol must be of importance. The introduction of only one hydroxy group in trans-stilbene produced potent inhibition of the I(Cl-,OR) (IC50: 3.3 microM). But in contrast, resveratrol with hydroxy groups at positions 4, 3', and 5' as well as methoxy substituted stilbene derivatives and 17beta-estradiol were ineffective. On the other hand, hexestrol potently inhibited I(Cl-,OR) indicating that the aromatic ring systems can also be connected by an ethyl bridge. In summary, a hydroxy group at position 4 (or 4') is a prerequisite for diethylstilbestrol-mediated inhibition of I(Cl-,OR).

Animals↗
PubMed1986

Possible role of oxygen radicals in cell transformation by diethylstilbestrol and related compounds.

Diethylstilbestrol (DES) and four derivatives, viz. tetrafluoro-DES, 3'-hydroxy-DES, Z,Z-dienestrol and hexestrol, were examined for their abilities to form superoxide radicals and to induce DNA strand breaks in the presence of horseradish peroxidase/hydrogen peroxide metabolism in a cell-free system. Furthermore, the induction of strand breaks by these compounds was tested in Syrian hamster embryo (SHE) cells in vitro. Formation of superoxide radicals could be demonstrated by reduction of nitro blue tetrazolium for DES but not for its derivatives. With isolated superhelical DNA, induction of strand breaks in the presence of Fe3+ was observed for DES, tetrafluoro-DES and 3'-hydroxy-DES, while hexestrol and Z,Z-dienestrol were ineffective. In SHE cells, alkaline elution technique showed that DNA strand breaks were induced by DES and all derivatives tested, although only at cytotoxic concentrations. It is concluded that DES, under conditions of peroxidative metabolism, can give rise to superoxide generation and DNA strand breaks, and that these events may play a role in the process of DES-induced cell transformation.

Animals↗
PubMed2002

Peritoneal lesions in guinea pigs treated with hormones.

Foci of peritoneal fibrosis frequently occur with ovarian neoplasms, especially those of epithelial type, usually in association with foci of metastatic tumor or implants. However, in the absence of the latter, the presence of fibrosis cannot be clearly explained. To find an explanation for this phenomenon, we investigated the development of hormone-dependent peritoneal changes in guinea pigs. Of the 59 guinea pigs studied, 49 received one of the following hormones or hormone analogs: estradiol, diethylstilbestrol (DES), testosterone, estrone, Megace, human chorionic gonadotropin, Norethin, clomiphene, luteinizing hormone, dienestrol, hexestrol, and tamoxifen, over a period ranging from 3 to 9 months. Ten guinea pigs received sterile water (control cases). Multiple peritoneal fibrous plaques 1 to 7 mm in greatest dimension were found in 5 of 7 guinea pigs that received estradiol, 6 of 8 treated with DES, and 2 of 2 that received hexestrol and estrone. The largest lesions (fibrous polyps up to 1.2 cm in greatest dimension) were seen in those that received estrone; however, this hormone was poorly tolerated, as two of the animals that received it died after 1 and 2 months, respectively. Because DES was the best-tolerated hormone, we administered it to five additional guinea pigs for 12 months. All five developed fibrous plaques; three of them also developed peritoneal fibroleiomyomas. We conclude that estrogens can induce the development of fibrous plaques in the peritoneum and when they are administered for a long period of time they also induce the development of smooth muscle nodules. This finding is relevant to understanding the pathogenesis of both fibrous lesions associated with ovarian tumors and peritoneal leiomyomatosis.

Animals↗
PubMed1997

Comparison of the ligand binding specificity and transcript tissue distribution of estrogen receptors alpha and beta.

The rat estrogen receptor (ER) exists as two subtypes, ER alpha and ER beta, which differ in the C-terminal ligand binding domain and in the N-terminal transactivation domain. In this study we investigated the messenger RNA expression of both ER subtypes in rat tissues by RT-PCR and compared the ligand binding specificity of the ER subtypes. Saturation ligand binding analysis of in vitro synthesized human ER alpha and rat ER beta protein revealed a single binding component for 16 alpha-iodo-17 beta-estradiol with high affinity [dissociation constant (Kd) = 0.1 nM for ER alpha protein and 0.4 nM for ER beta protein]. Most estrogenic substances or estrogenic antagonists compete with 16 alpha-[125I]iodo-17 beta-estradiol for binding to both ER subtypes in a very similar preference and degree; that is, diethylstilbestrol > hexestrol > dienestrol > 4-OH-tamoxifen > 17 beta-estradiol > coumestrol, ICI-164384 > estrone, 17 alpha-estradiol > nafoxidine, moxestrol > clomifene > estriol, 4-OH-estradiol > tamoxifen, 2-OH-estradiol, 5-androstene-3 beta, 17 beta-diol, genistein for the ER alpha protein and dienestrol > 4-OH-tamoxifen > diethylstilbestrol > hexestrol > coumestrol, ICI-164384 > 17 beta-estradiol > estrone, genistein > estriol > nafoxidine, 5-androstene-3 beta, 17 beta-diol > 17 alpha-estradiol, clomifene, 2-OH-estradiol > 4-OH-estradiol, tamoxifen, moxestrol for the ER beta protein. The rat tissue distribution and/or the relative level of ER alpha and ER beta expression seems to be quite different, i.e. moderate to high expression in uterus, testis, pituitary, ovary, kidney, epididymis, and adrenal for ER alpha and prostate, ovary, lung, bladder, brain, uterus, and testis for ER beta. The described differences between the ER subtypes in relative ligand binding affinity and tissue distribution could contribute to the selective action of ER agonists and antagonists in different tissues.

Amino Acid Sequence↗
PubMed1995

[Biosynthesis and microbial transformation of griseofulvin and carcinogenesis and prevention of cancer by estrogens].

In the biosynthetic study of griseofulvin by Penicillium urticae and microbial transformation of (-)- and (+)-dehydrogriseofulvin and their derivatives by Streptomyces cinereocrocatus excellent informations were obtained from 2H-NMR spectroscopy. In the reduction of (-)-dehydrogriseofulvin into (+)-griseofulvin by a partially purified enzyme system of S. cinereocrocatus, the origin of the 6' alpha-hydrogen of (+)-griseofulvin was a hydride ion donated by pro-4R-hydrogen of NADPH. In connection with the study of carcinogenesis, diethylstilbestrol (DES) was proved to disrupt microtubules in vitro. The other synthetic estrogens, E,E-dienestrol, meso-hexestrol, and dl-hexestrol were inhibitors of microtubule assembly in vitro, and induced twisted ribbon structures or ribbon-sheet-microtubules from microtubule proteins. Next, the effects of DES and its methyl ethers on the chromosome of and the cellular microtubule architecture, revealed by fluorescent anti-tubulin antibody, of Chinese hamster V79 cells were examined, and further estradiol-17 beta was proved to exhibit higher microtubule-disruptive activity than DES in V79 cells. Furthermore, cytoplasmic microtubules in the human breast cancer cell lines MCF-7 and MDA-MB-231, estrogen receptor-positive and -negative cell lines, respectively, were disrupted equally by estradiol-17 beta. Then, natural estrogens and their derivatives comprising 30 compounds in total were tested in Chinese hamster V79 cells, proving that 2-methoxyestradiol showed the strongest activity (EC50: 2 microM) to disrupt microtubules. Further, in the assay of indenestrol A, a metabolite of DES, indenestrol B and their monomethyl ethers, the 4'-methyl ether of [(-)-3S]-indenestrol B exhibited both the strongest cytotoxicity in, and greatest disruption of the cellular microtubules of V79 cells, and no correlation with the affinity for estrogen receptors was shown.

2-Methoxyestradiol↗
PubMed2006

HPLC-tandem mass spectrometric method to characterize resveratrol metabolism in humans.

BACKGROUND: Nutritional biomarkers are alternatives to traditional dietary assessment tools. We sought to develop a method for nutritional analysis of resveratrol, a phenolic compound with purported health-promoting properties, and to determine all resveratrol metabolites. METHODS: We obtained LDL and urine samples from 11 healthy male volunteers who had consumed 250 mL of Merlot red wine. We measured resveratrol and its metabolites with 96-well solid-phase extraction plates coupled with HPLC-tandem mass spectrometry. Hexestrol was used as the internal standard. Gradient chromatography in multiple reaction monitoring mode was performed on a Luna C(18) column, maintained at 40 degrees C; m/z transitions were as follows: resveratrol, 227/185; resveratrol glucosides, 389/227; resveratrol glucuronides, 403/227; resveratrol sulfates, 307/227; taxifolin, 303/285; and hexestrol, 269/134. RESULTS: Standard calibration curves were linear at 4.4-3289.5 nmol/L. Residual analyses were 100% (3.2) for trans-resveratrol and 100% (11.1) for trans-piceid. In both matrices, imprecision (CV) was <10.8% at all concentrations. Detection limits for resveratrol were 0.2 nmol/L (LDL), 0.3 nmol/L (synthetic urine), and 4.0 nmol/L (blank urine). Resveratrol and metabolites were checked for stability, and no degradation was observed. CONCLUSIONS: The HPLC-tandem mass spectrometry method enabled us to identify resveratrol sulfates in human LDL and to characterize the complete profile of resveratrol metabolism in human LDL and urine. This method provides an accurate index of exposure to resveratrol and its metabolites, which can be used as nutritional biomarkers for evaluating the biological effects of moderate wine intake on human health.

Adolescent↗