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This study evaluated the sorption and transport potential of seven phototransformation products of 17α-trenbolone, 17β-trenbolone, trendione, and altrenogest, along with the parent trienone steroids in batch and column soil-water systems. In batch systems, the target solutes exhibited linear isotherms, with values for sorption coefficients (log Koc) of parent steroids (2.46-2.76) higher than those for photoproducts (1.92-2.57). In column systems, the estimated retardation factors (Rsol) for parents (2.7-5.1) were ∼2-5 times higher than those for photoproducts (0.84-1.7). The log Koc (R2 = 0.75) and Rsol (R2 = 0.89-0.98) were well correlated with measured log Kow values, indicating that hydrophobic partitioning governed the soil-solute interaction of these biologically potent compounds in soil-water systems. These data indicated that photoproducts exhibited reduced sorption affinity and increased transport potential relative to more hydrophobic parent structures. In agroecosystems, traditional runoff management practices would be expected to exhibit reduced treatment effectiveness for photoproducts relative to the parent compounds of commonly used trienone steroids.

The detection of boldenone, nandrolone, 5(10)-estrene-3β,17α-diol, and 4-estrene-3,17-dione in a urine sample collected from a gelding having been treated with testosterone (500 mg 'Testosterone Suspension 100', single dose, injected intramuscularly) in 2009 led the authors' laboratory to suspect that these 'testicular' steroids could be minor metabolites of testosterone in geldings. Administration trials on six castrated horses with Testosterone Suspension 100 confirmed that low levels of boldenone, nandrolone, 5(10)-estrene-3β,17α-diol, and 4-estrene-3,17-dione could indeed be detected and confirmed in the early post-administration urine samples from all six geldings. Although boldenone has been reported to be present in urine after testosterone administration, there has been no direct evidence reported that boldenone, nandrolone, 5(10)-estrene-3β,17α-diol, and 4-estrene-3,17-dione are metabolites of testosterone in geldings. Subsequent in vitro experiments involving the incubation of testosterone with horse liver microsomes, liver, adipose and muscle tissues, and adrenal cortex homogenates failed to provide evidence that these four substances are minor metabolites of testosterone. An administration trial using 'Testosterone Suspension 100' supplemented with 13 C-labelled testosterone (500 mg, 1:1 ratio, injected intramuscularly) was performed. The similarities of the excretion curves of 12 C-testosterone and 13 C-testosterone in urine suggest that there was minimal kinetic isotope effect. 13 C-Labelled boldenone, nandrolone and 4-estrene-3,17-dione were detected but not 5(10)-estrene-3β,17α-diol and its 13 C-counterpart. This is the first unequivocal evidence of boldenone, nandrolone and 4-estrene-3,17-dione being metabolites of testosterone in geldings. In view of these results, caution should be exercised when interpreting findings of boldenone, nandrolone and/or 4-estrene-3,17-dione together with a relatively high level of testosterone in gelding urine. Copyright © 2017 John Wiley & Sons, Ltd., (Copyright © 2017 John Wiley & Sons, Ltd.)

Synovex® ONE is an extended-release implant containing the active ingredients estradiol benzoate and trenbolone acetate for use in beef steers and heifers. Trenbolone acetate is rapidly hydrolyzed in cattle to form 17β-trenbolone and its isomer, 17α-trenbolone, which are further transformed to a secondary metabolite, trendione. As part of the environmental assessment for the use of Synovex ONE, data were generated to characterize the fate of 17α-trenbolone, which is the principal metabolite found in cattle excreta, in the environment. A study was conducted to determine the degradation and transformation of [ 14 C]-17α-trenbolone in 2 representative water-sediment systems under aerobic conditions. The same transformation products, 17β-trenbolone and trendione, were formed, principally in the sediment phase, in both systems. From the production of these transformation products, the 50% disappearance time (DT50) values of 17β-trenbolone and trendione were determined, along with the DT50 values of the parent compound and the total drug (17α-trenbolone + 17β-trenbolone + trendione). The DT50 values for the total system (aqueous and sediment phase) and for the total residues (17α-trenbolone + 17β-trenbolone + trendione) in the 2 systems were 34.7 d and 53.3 d, respectively. Environ Toxicol Chem 2017;36:630-635. © 2016 SETAC., (© 2016 SETAC.)

Membrane-assisted solvent extraction (MASE) coupled to liquid chromatography-triple quadrupole mass spectrometry (LC-MS/MS) was studied for the determination of a variety of emerging and priority compounds in wastewater. Among the target analytes studied certain hormones (estrone (E1), 17β-estradiol (E2), androsterone (ADT), 17α-ethynyl estradiol (EE2), diethylstilbestrol (DES), equilin (EQ), testosterone (TT), mestranol (MeEE2), 19-norethisterone (NT), progesterone (PG) and equilenin (EQN)), alkylphenols (APs) (4-tert-octylphenol (4tOP), nonylphenol technical mixture (NPs) and 4n-octylphenol (4nOP)) and BPA were included. The work was primarily focused in the LC-MS/MS detection step, both in terms of variable optimization and with respect to the matrix effect study. Both, electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) were assessed both in the negative and positive mode, including the optimization of MS/MS operating conditions. The best results were obtained, in most of the cases, for ESI using 0.05% ammonium hydroxide as buffer solution in the mobile phase, composed with methanol and water. Under optimum detection conditions, matrix effect during the detection step was thoroughly studied. Dilution, correction with deuterated analogues and clean-up of the extracts were evaluated for matrix effect correction. Clean-up with Florisil together with correction with deuterated analogues provided the most satisfactory results, with apparent recoveries in the 57-136% range and method detection limits in the low ngL(-1) level for most of the analytes. For further validation of the method, two separated extraction procedures, the above mentioned MASE, and conventional solid phase extraction (SPE) were compared during the analysis of real samples and comparable results were successfully obtained for E1, E2, EE2, DES, NT, TT, EQ, PG, BPA, ADT, 4nOP, 4tOP, NPs and EQN., (Copyright © 2014 Elsevier B.V. All rights reserved.)

In vitro assays are broadly used tools to evaluate the estrogenic activity in Waste Water Treatment Plant (WWTP) effluents and their receiving rivers. Since potencies of individual estrogens to induce in vitro and in vivo responses can differ it is not possible to directly evaluate risks based on in vitro measures of estrogenic activity. Estrone, 17beta-estradiol, 17alfa-ethinylestradiol and to some extent, estriol have been shown to be responsible for the majority of in vitro estrogenic activity of municipal WWTP effluents. Therefore, in the present study safe concentrations of Estrogenic Equivalents (EEQs-SSE) in municipal WWTP effluents were derived based on simplified assumption that the steroid estrogens are responsible for all estrogenicity determined with particular in vitro assays. EEQs-SSEs were derived using the bioassay and testing protocol-specific in vitro potencies of steroid estrogens, in vivo predicted no effect concentration (PNECs) of these compounds, and their relative contributions to the overall estrogenicity detected in municipal WWTP effluents. EEQs-SSEs for 15 individual bioassays varied from 0.1 to 0.4ng EEQ/L. The EEQs-SSEs are supposed to be increased by use of location-specific dilution factors of WWTP effluents entering receiving rivers. They are applicable to municipal wastewater and rivers close to their discharges, but not to industrial waste waters., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

In this study we applied DIMETRIS (dimethyl(3,3,3-trifluoropropyl)silyldiethylamine), a new silylating reagent, to derivative natural estrogens such as estrone (E1), 17β-estradiol (E2) and estriol (E3), as well as the synthetic 17α-ethinylestradiol (EE2) and the non-steroid diethylstilbestrol (DES). Its derivatizing properties were compared with those of the commonly used mixture of BSTFA (N,O-bis(trimethylsilyl)trifluoroacetamide)+1% trimethylchlorosilane (TMCS) and with MTBSTFA (N-tert-butyldimethylsilyl-N-methyltrifluoroacetamide). The use of DIMETRIS for the silylation all of them is reported for the first time. The nucleophilic properties of DIMETRIS were found to be superior to those of MTBSTFA, but slightly inferior to those of BSTFA. It was used to derivatize steroid (E1, E2, E3 and EE2) and non-steroid (DES) estrogens at 30°C prior to GC/MS analysis. These DMTFPS-derivatives exhibited good separation (low retention times despite the high molecular masses) and ionization properties in GC/MS analyses (the highest relative response factors for DMTFPS-derivatives among those tested). However, DIMETRIS and MTBSTFA (which produce mono-O-silyl derivatives of EE2) should not be used for the simultaneous analysis of EE2 and E1. Only a mixture of BSTFA+1% TMCS in pyridine, which generates the fully derivatized EE2 product (stable in GC injector), permits the determination of these two estrogenic compounds during one GC-MS run. On the other hand, because DIMETRIS requires a lower derivatization temperature than BSTFA, it could be very useful for the derivatization of thermally unstable estrogenic compounds. In the next step of this study, the SPE-GC-MS method based on DIMETRIS derivatization for the analysis of DES, E2 and E3 in aqueous samples was evaluated and validated. The MQL values: 1.4, 1.6 and 1.5ngL(-1) for DES, E2 and E3, respectively, proved its suitability to determine target compounds in environmental samples. Finally, the proposed method was successfully applied to the analysis of selected estrogenic compounds in real seawater and wastewater samples in Poland., (Copyright © 2013 Elsevier B.V. All rights reserved.)

A molecularly imprinted polymer was synthesized by ultrasonic irradiation, with attapulgite as matrix using β-naphthol as the template molecule, acryloyl-β-cyclodextrin as the functional monomer, and N,N-methylenebiacrylamide as the cross-linking agent, respectively. The imprinted polymer was characterized by infrared spectroscopy and transmission electron microscopy. Compared to polymers prepared by traditional heat sources, the molecularly imprinted polymer synthesized by ultrasonic irradiation had better selectivity and faster adsorption kinetics to estriol, estradiol, estrone and diethylstilbestrol. Using the imprinted polymer as the packing material for on-line solid-phase extraction, the above four estrogens in milk samples were concentrated and analyzed. The limits of detection for these estrogens were in the range of 1-8 ng g(-1) and reproducibility were less than 5.1% as RSDs (n=6) with milk samples spiked at 100 and 1000 ng g(-1) of each analyte., (Crown Copyright © 2012. Published by Elsevier B.V. All rights reserved.)

Manure is increasingly being viewed as a threat to aquatic ecosystems due to the introduction of natural and synthetic hormones from land application to agricultural fields. In the Midwestern United States, where most agricultural fields are tile-drained, there is little known about hormone release from fields receiving animal wastes. To this end, seven sampling stations (four in subsurface tile drains and three in the receiving ditch network) were installed at a Midwest farm where various types of animal wastes (beef, dairy, and poultry lagoon effluent, dairy solids, and subsurface injection of swine manure) are applied to agricultural fields. Water flow was continuously monitored and samples were collected for hormone analysis during storm events and baseline flow for a 15 month study period. The compounds analyzed included the natural hormones 17α- and 17β-estradiol, estrone, estriol, testosterone, and androstenedione and the synthetic androgens 17α- and 17β-trenbolone and trendione. Hormones were detected in at least 64% of the samples collected at each station, with estrone being detected the most frequently and estriol the least. Testosterone and androstendione were detected more frequently than synthetic androgens, which were detected in fewer than 15% of samples. Hormone concentrations in subsurface tile drains increased during effluent irrigation and storm events. Hormones also appeared to persist over the winter, with increased concentrations coinciding with early thaws and snowmelt from fields amended with manure solids. The highest concentration of synthetic androgens (168 ng/L) observed coincided with a snowmelt. The highest concentrations of hormones in the ditch waters (87 ng/L for total estrogens and 52 ng/L for natural androgens) were observed in June, which coincides with the early life stage development period of many aquatic species in the Midwest.

To study the roles of estrogens and estrogen metabolites (EMs) in breast carcinogenesis, we reported a quantitative liquid chromatography-tandem mass spectrometry (LC-MS/MS) method utilizing selective reaction mode (SRM) to analyze estrogens and EMs in the extracellular and intracellular compartments of endogenous MCF-7 breast cancer cells through simple ethyl acetate (EA) extraction and dansyl chloride derivatization. Under a 35-min LC gradient elution on a reversed phase C18 column, the method was shown to simultaneously quantify 12 estrogens and EMs: estrone (E1) and its 2-, 4-, 16α-hydroxy derivatives (2-OHE1, 4-OHE1, 16α-OHE1), and 2-, 4-methoxy derivatives (2-MeOE1, 4-MeOE1); 17β-estradiol (E2) and its 2-, 4-hydroxy derivative (2-OHE2, 4-OHE2) and 2- and 4-methoxy derivatives (2-MeOE2 and 4-MeOE2); and estriol (E3), using ethinylestradiol (EE2) as the internal standard (IS). Using a calibration curve-standard addition hybrid method, we were able to determine the amount of estrogens and EMs in not only the treated cells but also the non-treated cells. The limits of quantification (LOQs) were determined to range from 0.05-80 pg on column with an inter-batch accuracy around 72-123% and precision around 1-10%. Results indicated that trace amounts (<0.9 fg/cell) of E1 and E2 were present in both the extra- and intra-cellular compartments under non-treated condition but DMSO could induce E1 and E2 as well as trace amounts (<2.25 fg/cell) of EMs in the cell. E2 treatment substantially increased not only E1 and E2 in the intra-cellular (60 fg/cell) and extra-cellular (3000 fg/cell) compartment but also substantially induced EMs primarily in the extracellular compartment (0.6-25 fg/cell). These data implied that EMs could be quickly generated and distributed to the extracellular compartment by E2 within 24h of treatment and DMSO solvent could potentially induce slight estrogen effects., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Trenbolone acetate (TBA) is a synthetic androgenic steroid hormone administered as a subcutaneous implant for growth promotion in beef cattle. The primary metabolite excreted in manure from implanted cattle is 17alpha-trenbolone with lesser amounts of 17beta-trenbolone and trendione also present. At 22 degrees C and favorable moisture conditions in a controlled laboratory environment, trenbolone degrades to trendione in a few hours; however, these conditions are often not what exist in the field. Therefore, aerobic degradation rates of 17alpha-trenbolone, 17beta-trenbolone and trendione were determined in a sandy soil and silty clay loam under a range of temperature and water availability combinations that may be expected in the field. A first-order exponential decay model was used to estimate rates and generally resulted in good model fits to the data. Degradation rates decreased with decreasing water availability (i.e., more negative soil matric potential) and decreasing temperature. However, when water availability was substantially reduced (-1.0MPa), hotter temperatures (35 degrees C) significantly reduced trenbolone degradation rates. Once temperature was low enough to limit microbial activity, no further changes were observed with decreasing matric potential. Trendione also exhibited similar moisture and temperature dependent degradation, but persisted longer than the parent trenbolone. The latter was discussed in light of extracellular versus intracellular enzymatic degradation and sorption. Half lives at colder temperatures (5 degrees C) even under favorable moisture conditions were 2-3d for the trenbolone isomers and approached 10d for trendione.

For almost two decades we have known that enzymatic hydrolysis of "normal" urine samples from the entire male horse using Escherichia coli (E. coli) followed by solvolysis (ethyl acetate:methanol:sulphuric acid) results in the detection of significant amounts of estr-4-ene-3,17-dione (19-norandrost-4-ene-3,17-dione) along with estr-4-en-17beta-ol-3-one (19-nortestosterone, nandrolone) in extracts of the hydrolysed urine and that both steroids are isolated from the solvolysis fraction. This solvolysis process is targeted at the steroid sulphates. Also we have shown that 19-norandrost-4-ene-3,17-dione and 19-nortestosterone are isolated from testicular tissue extracts. Subsequently, evidence was obtained that 19-nortestosterone detected in extracts of "normal" urine from male horses may not be derived from the 17beta-sulphate conjugate. However, following administration of 19-nortestosterone based proprietary anabolic steroids to all horses (males, females and castrates), the urinary 19-nortestosterone arising from the administration is excreted primarily as the 17beta-sulphate conjugate. Thus, if the 19-nortestosterone-17beta-sulphate conjugate arises only following administration this has interesting implications for drug surveillance programmes to control administration of 19-nortestosterone based anabolic preparations to male horses. These results have led us to consider that the precursors to 19-nortestosterone and 19-norandrost-4-ene-3,17-dione, present in the urine prior to the hydrolysis steps, have the same basic structure except for the functionality at the 17-position. We have used preparative high pressure liquid chromatography (LC) and LC fractionation to separate these precursors from the high amounts of oestrogenic sulphates present in "normal" urine from the entire male horse. Purified fractions have then been studied by liquid chromatography-mass spectrometry (LC-MS) and gas chromatography-mass spectrometry (GC-MS) to identify the precursors.

A novel method of micro-plate magnetic chemiluminescence enzyme immunoassay (MMCLEIA) for the screening of 17beta-estradiol in water samples was proposed. It used the micro-plate magnetic separator designed by ourselves which can achieve the high-throughput analysis without the samples pre-treatment and the sensitive chemiluminescence system of AMPPD-ALP system. The method showed specific recognition of estrogen, without cross-reactions for three other major estrogenic compounds (17beta-estradiol (E2), estriol (E3), ethinyl (E2)) commonly found in water. The MMCLEIA was also especially suitable for the large-scale samples processing. The working range for 17beta-estradiol was 10-3000 pg/ml. The assay sensitivity was 5.4 pg/ml. Both intra- and inter-assay had relative standard deviation of less 15%. The effect of several physico-chemical parameters, such as the ratio of antibody versus antigen, incubation time and the concentration of detergent were studied. This method has been successfully applied to the preliminary detection of the sea-water. Compared with the chemiluminescence enzyme-linked immunosorbed assay, the correlation was good.

The aqueous chlorination kinetics of six endocrine disruptors (EDs: 4-n-nonylphenol, beta-estradiol, estrone, estriol, 17alpha-ethinylestradiol, progesterone) were studied in the 3.50-12.00 pH range, at 20+/-2 degrees C, in the presence of an excess of total chlorine. Under these conditions, all molecules with a phenolic group in their structure were rapidly oxidized by chlorine, whereas progesterone remained unchanged. In the first step, apparent kinetic rate constants were determined at various pH levels. Then each elementary reaction kinetic rate constant, i.e., the reaction of hypochlorous acid (HOCl) with ionized EDs and neutral EDs and an acid-catalyzed reaction of HOCl with neutral EDs, was calculated in the second step. The results showed that chlorination exhibits a second-order reaction rate. The rate constants for the acid-catalyzed reaction ranged from 3.02 x 10(4) M(-1) s(-1) (for 4-n-nonylphenol) to 1.82-2.62 x 10(5) M(-1) s(-1) (for hormones). The rate constants of HOCI reactions with ionized EDs were found to be equal to 7.5 x 10(4) M(-1) s(-1) (for 4-n-nonylphenol) and between 3.52 and 4.15 x 10(5) M(-1) s(-1) (for hormones), while the rate contants of HOCI with neutral EDs were much lower, i.e., between 1.31 M(-1) s(-1) (for 4-n-nonylphenol) and 3.74-4.82 M(-1) s(-1) (for hormones). At pH 7, the apparent-second-order rate constants were calculated to range from 12.6 to 131.1 M(-1) s(-1). For a total chlorine concentration of 1 mg/L, the corresponding half-life times at pH 7 were about 65 min for 4-n-nonylphenol and 6-8 min for hormones.

Xenobiotic and endobiotic molecules can react with DNA leading to formation of so-called DNA adducts. This modified DNA can be repaired enzymatically, but, if not, these modifications are believed to be responsible for the initiation of carcinogenic processes. Hence, we studied the interaction of 2'-deoxynucleosides and 2'-deoxynucleotides with 3,4-estronequinone (3,4-E(1)Q), a metabolite of estrone (E(1)) and a supposed carcinogen. These estrone-nucleic acid adducts were analysed by capillary liquid chromatography (CapLC) coupled to electrospray ionization mass spectrometry (ESI-MS). Knowledge of their behaviour from in vitro studies is a prerequisite for detecting adducts in in vivo studies. Our initial attempts to synthesise nucleos(t)ide adducts of 3,4-E(1)Q in an aprotic solvent (dimethylformamide) yielded no adducts. However, under acidic aqueous conditions, adducts were obtained. With dGuo, a dGuo adduct was found in addition to a Gua adduct. Earlier publications on adduct formation in protic solvents failed to report formation of any adduct with dAdo. A N(3)-Ade adduct was reported upon reaction of 3,4-E(1)Q with Ade base and with DNA. With dAdo, we obtained two nucleoside adducts and six Ade adducts due to loss of 2'-deoxyribose. Thus, contrary to general belief that only 2,3-E(1)Q can form stable adducts, we showed formation of substantial amounts of intact DNA adducts with 3,4-E(1)Q in addition to deglycosylated adducts. Adducts were also obtained with dGMP and dAMP, but no phosphate alkylation was found. Adducts of dCyd, dCMP, dThd, and dTMP were not detected. Using chromatographic-MS data a structural relationship between the 2'-deoxynucleoside, 2'-deoxynucleotide and base adducts was found in the various reaction mixtures. The adducts of dGuo and dGMP reaction mixtures were alkylated at the same N(7)-position of the nucleobase, as indicated by the occurrence of a rapid deglycosylation reaction. In dAdo and dAMP reaction mixtures, 14 adducts were detected; their relationships from the LC and MS data reduced the number of structures to six adenine base alkylated adducts with respect to alkylation between N(1), N(3), N(7) and/or N(6) in the adenine and C(1), C(2) and/or C(6) in 3,4-E(1)Q. We could infer, in addition, whether they had an A ring attachment or a C(6) attachment on the estrone moiety., (Copyright 2004 John Wiley & Sons, Ltd.)

The title compound 17 has been synthesized for the use as hapten in the development of a competitive enzyme immunoassay for estrogen sulfamates. The synthesis started from estradiol diacetate 2. Oxyfunctionalization at C-11 to give 11alpha-hydroxy steroid 8 was accomplished by hydroboration/alkaline hydrogen peroxide oxidation of the 9(11)-dehydro derivative 7, which was obtained from compound 2 via 9-hydroxylation with dimethyldioxirane. After transformation of compound 8 into the allyl ether 9, the side chain was thio-functionalized at the omega-position affording the thioate 11 in two steps. Selective silylether deprotection at position 3 followed by sulfamoylation gave the sulfamate 19, which in turn was demasked at position 17 and treated with sodium borohydride/aluminum chloride to liberate the side chain thiol. Alternatively, title compound 17 was synthesized via the disulfides 13-16. For the preparation of the immunogen the title compound 17 was coupled to bovine gamma globulin in a two-step procedure using an amine and thiol specific bifunctional crosslinker. The immunization of rabbits resulted in the formation of antibodies which clearly discriminated the sulfamoylated estrogens from the non-esterified estrogens. The use of a biotinylated hapten derivative as a tracer in combination with a streptavidin-peroxidase-tetramethylbenzidine based detection system allowed the measurement of estradiol 3-sulfamate (1) in the range of about 1 to 1000 pg/well.

Factors affecting the validity and interpretation of androgen receptor (AR) assays on prostatic carcinoma tissue are discussed, and attempts to relate AR concentration with response of the patient to hormonal manipulation are reviewed. It is concluded that nuclear AR content may be useful in evaluating prognosis and likely response to hormonal therapy, and that, owing to the heterogeneity of many prostatic carcinoma specimens, a reliable histochemical method for the assessment of androgen sensitivity would be a valuable supplement to the biochemical assay.

Certain previously unrecognized ligands of SHBG of low polarity in pregnancy were identified. They include two weakly bound compounds: 5 alpha-pregnane-3,20-dione and progesterone; and two strongly bound substances, 2-methoxyestrone and a new steroid, estradienolone (17 beta-hydroxy-1,5-estradiene-3-one). The identification of the first three peaks was based on chromatographic elution patterns, binding characteristics and gas chromatography-mass spectrometry. The identification of the fourth peak, the new steroid, was based on similar kinds of evidence and, in addition, solubility characteristics and ultraviolet absorption spectrum.

Analytical methodology is described for determining residues of the synthetic anabolic steroid trenbolone acetate (TBA) and its hydrolysis product trenbolone (TBOH) in admixture in animal chow, human urine, and wastewater. Benzene extracts of the substrates are subjected to liquid-liquid partitioning, further cleanup on a column of silica gel, and direct analysis by high pressure liquid chromotography or derivatization with pentafluoropropionic anhydride and analysis by electron capture gas chromatography. Satisfactory recoveries were obtained with both compounds from all three substrates. Residue levels of TBA and TBOH as low as 0.32 and 0.04 ppm, respectively, could be detected in chow; about 0.6 ppb of each compound could be detected in urine and wastewater. Thin layer chromatographic behavior of the two compounds in 7 solvent systems and other ancillary analytical data are also presented.

The crystal and molecular structure of 3-methoxy-9(10 leads to 19)abeo-1,3,5(10)-estratrien-17-one has been determined by X-ray analysis in order to ascertain the configuration at C(9). The seven-membered B-ring has a chair conformation and this causes the molecule to bow towards the alpha-face rather than towards the beta-face as in estradiol.

Anabolic agents applied in animal production can be classified as sex hormones, as far as one aspect of their biological activity is concerned. In respect to their chemical nature the agents can be divided into three sub-groups: (a) steroids natural to the body (endogenous steroids); (b) steroids foreign to the body; (c) other compounds foreign to the body. After exogenous application (oral or parenteral) metabolism of compounds in group (a), e.g. 17beta-estradiol or testosterone, follows the pathways for the identical endogenous hormones; i.e. enzymatic transformation of the biologically active molecule into less active compounds. Excretion seems to occur predominantly via the feces, followed by the excretion via the urine. Steroids foreign to the body (group [b]) may be simple esters of endogenous steroids (estradiol benzoate, testosterone propionate) or compounds with basically modified steroidal structures, like trenbolone acetate (estra-4, 9, 11-trien-17beta-OH-3one-Ac). After enzymatic cleavage of an ester in the animal, metabolism of natural steroids again follows the endogenous pathways. Other steroids are mainly excreted without changes of the original steroid structure. It has been demonstrated, that compounds applied as esters have a delayed elimination. Anabolic agents of nonsteroidal structure, (group [c]) like stilbene derivatives and zeranol (6-[6, 10-dihydroxy-undecyl] beta-resorcylicacid-mu-lactone) are not, or only to a minor percentage, catabolized in the body. It can be generalized so far, that the fraction of the hormone eliminated in the urine, is in the conjugated from, while the fraction appearing in the feces is in the free form. This is due to enterohepatic circulation, which occurs with both, the intact or catabolized compound. The elimination rate of anabolic agents not only depends on the absorption after oral or parenteral application, which is strongly related to the type of formula---or cleavage of possible esters, but also on the metabolic clearance rate, which is a function of several factors, for example binding to plasma proteins or tissue structures. Differences in the elimination rate between compounds could be attributed to these phenomena. Tissue residue formation not only depends on the elimination rate, but also on other factors like mode of application, formula and withdrawal time. The possibility for their detection is related to the method applied. In cases of adequate treatments of the animals, and with the exception of the implantation site (see below), positive hormone values in tissues (qualitative and quantitative) could only be obtained, when radioimmunoassay techniques with a sensitivity in the low nanogram range were applied...

A technique of high-performance thin-layer chromatography (HPTLC plates) by which to identify the anabolic substance of trenbolone acetate (TBA) and its metabolite trenbolone is described in this paper. Plasma, bile, urine, faeces, liver, and meat can be used for testing. The sensitivity of the method is 1 ng. Additional reliability criteria are enumerated. Semiquantitative TBA determination has proved to be suitable in random sampling for residual analysis.

Identification and quantitation of trace amounts of trenbolone in bovine tissue by capillary gas chromatography-mass spectrometry-selected-ion monitoring (GC-MS-SIM) has been developed. Three-phase liquid-liquid extraction using a mixture of water-acetonitrile-dichloromethane-hexane was utilized for the sample extraction from tissue. Target compounds were extracted from the tissue into the acetonitrile layer. The residue from this extraction was then subjected to solid-phase extraction by C18 and silica gel disposable cartridges using methanol-water and benzene-acetone as eluents. To overcome extensive matrix interferences, preparative reversed-phase high-performance liquid chromatographic separation was used with an octadecyl-bonded column using methanol-water as mobile phase for sample clean-up prior to GC-MS analysis. A structural analogue of trenbolone, 19-nortestosterone, was chosen as the internal standard for quantitation by GC-MS. The sample was co-injected with N,O-bis (trimethylsilyl) trifluoroacetamide-1-(trimethylsilyl) imidazole (95:5, v/v) for flash heater derivation. Identification and quantitation were simultaneously carried out by SIM of characteristic ions of the trimethylsilyl derivatives of trenbolone and 19-nortestosterone. The limit of detection for trenbolone and epitrenbolone was 0.5 ppb in muscle and liver tissue. A comparison of sensitivity and specificity between GC-MS under electron ionization in addition to positive- and negative-ion chemical ionization conditions using methane reagent gas is also discussed.

Estradiol (ER), progesterone (PR), androgen (AR) and glucocorticoid receptor (GR) levels were assayed in 25 breast cancer tumors. The tissue was pulverized and homogenized in buffer, then divided into two parts: one was assayed by the standard dextran-coated charcoal method (DCC), with Scatchard plot analysis, the other was assayed by a micromethod developed in our laboratory, as described below: --incubation of the cytosol with several ligands (labelled and unlabelled) selected to avoid unwanted cross-reactions --DCC separation, followed by extraction of all receptor-bound steroids by precipitation of proteins with methanol/TCA --separation of these steroids on a high pressure liquid chromatography (HPLC) column using a methanol/water solvent --collection of the fractions of the column outlet and counting. Use of three labelled ligands and appropriate unlabelled ligands allowed assays of the four receptors. This micromethod was highly correlated with the standard method: ER = 0.985 (P less than 0.001); PR = 0.999 (P greater than 0.001); AR = 0.989 (P less than 0.001); GR = 0.867 (P less than 0.001). Thresholds of positivity were not modified. This micromethod allowed simultaneous measurement of several receptors in 40 mg biopsy specimens and can be applied to other hormone-dependent tissues.

Using high-pressure liquid chromatography (HPLC) the antiprogestin RU 486 and two of its metabolites (N-monodemethyl RU 486 and propargyl RU 486) were measured in plasma and follicular fluid of 21 women requesting laparoscopic sterilization. Pretreatment of the women involved ovulation induction with clomiphene and HCG. RU 486 (100 mg) was administered orally and 1 h later blood samples were withdrawn. Thirty-four hours later, at laparoscopy, samples of both blood and follicular fluid were collected. During the 34-h period the average plasma level of RU 486 decreased from 1.93 mumol/l to 0.91 mumol/l, i.e. by -50%. The latter concentration of RU 486 was not significantly different from that found in follicular fluid (0.79 mumol/l). The monodemethyl metabolite exhibited significantly higher plasma levels (3.09 mumol/l) than RU 486 1 h after administration. Thirty-four hours later these levels had decreased to 0.92 mumol/l, i.e. by 70%. In follicular fluid, the levels of the monodemethyl metabolite (1.76 mumol/l) were significantly higher than those of RU 486 (0.79 mumol/l). Because of background noise, only approximate values were established for the propargyl metabolite. These were 0.67 and 0.40 mumol/l, respectively, in plasma and 0.42 mumol/l in follicular fluid. The results indicate that RU 486 and two of its major metabolites can readily cross the blood-follicle barrier of human pre-ovulatory follicles.

Progesterone bound with high affinity to the endometrial and myometrial cytosol of ovariectomized bonnet monkeys pretreated with estradiol benzoate and progesterone. The equilibrium dissociation constant (Kd) of 3H-progesterone was 4.5 nM and 5.5 nM and the binding capacity was 1.7 nM and 1.4 nM for the endometrial and myometrial receptors, respectively. This experimental 'model' was used to assess the relative binding affinity (RBA) of progesterone, ZK 98.299 and ZK 98.734. The tested compounds showed competitive binding to cytoplasmic progesterone receptors. The RBA of progesterone in the endometrium (100) was more than that of ZK 98.299 (25.1) and ZK 98.734 (17.8). A similar RBA pattern was observed in the myometrial cytosol. Both ZK 98.299 and ZK 98.734, like progesterone, displaced the 3H-progesterone bound to the receptors. The administration of ZK 98.299 or ZK 98.734, during the mid-luteal phase, has been reported to shorten the cycle length in bonnet monkeys and marmosets, respectively. These compounds, therefore, appear to intercept the progesterone action by blocking progesterone binding sites in the target tissue. Since ZK 98.299 has higher binding affinity than ZK 98.734, it may be a more potent progesterone antagonist.

A new method for the qualitative and quantitative determination of trenbolone and testosterone residues in meat, liver, and kidney is described. The analytical procedure consists of the following steps: homogenisation of the meat sample with tetrahydrofurane; liquid-liquid partition first between acetonitrile and hexane, then between sodium hydroxide and petroleum ether/benzene; purification of a silica gel column. The purified sample is analysed by high pressure liquid chromatography using silica gel packings. Detection and quantitative determination are performed with a fluorescence and/or a spectrophotometric detector. Fluorescence measurements are carried out applying a self-made silica gel packed flow-cell. The detection limit in meat extracts is 50 ppb for testosterone and 5 ppb for trenbolone.

The development of a competitive microtitration plate enzyme-immunoassay for monitoring trenbolone application to animals is described. 'Bridge heterology' was achieved with a rabbit antibody raised against 17 beta-trenbolone-hemisuccinate-BSA and 17 alpha-trenbolone glucuronide-alkaline phosphatase as a tracer. The required 17 alpha-trenbolone glucuronide was prepared by application of trenbolone acetate to a calf following isolation from urine by three HPLC steps. The glucuronide was linked to alkaline phosphatase by the mixed anhydride procedure. The EIA was performed by coating affinity purified sheep IgG antirabbit IgG to the microtitration plate well followed by the addition of sample, tracer and hormone-specific antibody. The absolute detection limit was less than 1 pg; 50% relative binding at approximately 3 pg which is about 20 times superior to our RIA. The sample blanks of purified extracts from urine, bile, faeces, liver and muscle were similar in both methods (EIA and RIA) and much lower than required for a reliable detection of positive samples. Assay variations were always less than 15%. For the EIA, radiolabelled trenbolone, which is not commercially available, is not necessary and according to our experience the EIA is more practicable and economic.

Primary endometrial carcinomas from 35 non-treated patients were investigated by measurement of androgen receptors in the tumor cytosols. Receptor analysis was done with a labelled synthetic androgen, methyltrienolone 3H-R1881 and triamcinolone acetonide, and dextran-coated charcoal absorption. The concentrations of androgen receptors in the endometrial carcinomas were, in decreasing order: highly-differentiated tumors, 15.7 +/- 1.8 (fmol/mg protein, mean +/- SE) (number of cases, 21); moderately differentiated tumors, 4.6 +/- 1.6 (n = 7); poorly differentiated tumors, undetectable in 7 out of 8 cases. Highly-differentiated tumors contained a much greater concentration of receptors than the two less differentiated ones. One highly-differentiated endometrial carcinoma with pyometra virtually lacked the receptor. The moderately differentiated endometrial carcinomas contained very low levels of the receptors. The poorly differentiated tumors virtually lacked the receptors. Metastatic lymph nodes from primary endometrial carcinomas with moderate differentiation had a very low receptor level. From these results, it is concluded that human endometrial carcinomas, particularly with histologically high differentiation, contain a considerable amount of androgen receptor and that the receptor concentrations appear to correlate with the histologic grade of tumor differentiation.

After incubation of 1,3,5(10),16-oestratetraenol, a 16-dehydrosteroid, with rat liver microsomes, 16 alpha, 17 alpha-epoxy-oestratrienol was isolated as metabolite. The compound was detected by the use of mass fragmentography after purification of the incubation extract by thin-layer chromatography. Since the epoxide is rapidly hydrolysed by a hepatic epoxide hydratase, only very small concentrations of this metabolite were present in the incubation extract. When styrene oxide was added to the incubation mixture as inhibitor of the epoxide hydratase, the yield of the steroid epoxide increased considerably. Final identification of the oestrogen epoxide was performed by recording mass spectra and by comparison with authentic reference material.

The concentrations of RU 486 and its demethylated metabolites were determined by RIA in samples of myometrium, abdominal adipose tissue, and serum, which were collected at hysterectomies performed 12-15 h after oral administration of 200 mg RU 486. The RU 486 concentrations in myometrium were similar in the five women studied, with a mean of 148 +/- 58 (+/- SD) ng/g (344 +/- 135 pmol/g). The adipose tissue RU 486 levels varied more, the mean concentration being 447 +/- 191 ng/g (1041 +/- 445 pmol/g). The serum RU 486 concentrations ranged from 175-899 ng/ml [mean, 396 +/- 259 ng/mL (922 +/- 603 nmol/L)]. In these women the nonprotein-bound fraction of [6,7-3H]RU 486 varied from 1.4-3.1% (mean, 2.3%). The approximate concentrations of the combined mono- and didemethylated metabolites of RU 486 were 1.4, 3.1, and 5.2 times higher in adipose tissue, myometrial tissue, and serum, respectively, than those of the parent RU 486. In vitro, rapid and nonsaturable accumulation of [6,7-3H]RU 486 from phosphate buffer into adipose tissue was inhibited by the addition of alpha 1-acid glycoprotein, the specific serum transport protein for RU 486, to the buffer medium. Accumulation of [6,7-3H]RU 486 in myometrial specimens was poor. The enterohepatic cycling of RU 486 was assessed in four normal subjects by repetitive intake of charcoal subsequent to ingestion of 200 mg RU 486. Compared to other normal subjects, the serum levels and areas under the concentration curves were lower and t1/2 values shorter in the group given charcoal, suggesting that in vivo RU 486 may be partly pooled in the enterohepatic cycle. Our studies suggest that despite the low volume of distribution and the effective serum binding of RU 486, the myometrial and adipose tissue concentrations of RU 486 and its metabolites were similar (approximately 10(-9)-10(-10) mol/g) after oral intake of RU 486.

Comparison of the plasma levels of steroids between ovarian and peripheral venous blood in polycystic ovarian disease revealed that polycystic ovaries secreted androstenedione, testosterone, dihydrotestosterone, dehydroepiandrosterone and 17-hydroxyprogesterone significantly more than healthy ovaries at the early follicular phase of the cycle, whereas the levels of estradiol and progesterone did not differ from the control values. Follicular cyst fluid from polycystic ovaries showed significantly higher concentrations of testosterone, androstenedione jand dehydroepiandrosterone and much lower levels of estrone, estradiol, progesterone and 17-hydroxyprogesterone than follicular fluid from healthy preovulatory follicles. Testosterone and estradiol did not, however, show any significant difference when follicles in polycystic ovaries were compared to those in healthy ovaries at early follicular phase of the cycle. According to these findings follicle development in polycystic ovaries is arrested at a stage corresponding to the early follicular phase of the cycle. Increased androgen and 17-hydroxyprogesterone secretion by polycystic ovaries seems to originate mainly from the hyperplastic theca interna cells.

The current methods for the control of the illegal use of anabolics in cattle breeding are far from satisfactory in terms of specificity and sensitivity. Here we describe a method for the simultaneous detection of diethylstilbestrol, zeranol and trenbolone in plasma and edible cattle tissues. The method is rapid (total analysis time 30 min), specific and sensitive (ppt level). The technique employed is a tandem mass spectrometric method: collisionally activated decomposition mass-analysed ion kinetic energy (CAD MIKE) spectrometry. Detection involves the injection of the crude ethanol extracts of plasma and tissues and selection/collision of molecular ions of each targeted compound. Confirmation of the presence of particular compounds is made by matching the complete CAD spectra so obtained with those of the pure compounds.

An new method for the qualitative and quantitative determination of trenbolone residues in meat, liver and kidney is described. The analytical procedure consists of the following steps: comminution of the meat sample; homogenisation with tetrahydrofurane; liquid-liquid partition first between acetonitril and hexane then between sodium hydroxide solution and petroleum ether/benzene; chromatography on a silica gel column. The detection on the TLC-plate is performed with 366 nm ultra violet light. The quantitative determination is carried out by means of a remission spectral fluorimeter directly on the plate. The detection limit in meat extracts was 5 ppb.

A specific, sensitive, precise and accurate radioimmunoassay has been developed for the quantitation of the synthetic anabolic steroid trienbolone acetate (TBA) and its major metabolites, free and conjugated trienbolone (TBOH) in bovine tissues and plasma. With the extraction procedure described unspecific interference with the antigen-antibody reaction could be ruled out. The assay can significantly detect amounts of more than 40 pg TBOH and 70 pg TBA. 0.1 - 2.0 g tissue and 0.1 - 1 ml plasma are sufficient for 1 determination. Residues (range 0.1 -2.0 ng/g) were still present in calves 69 days after implantation of "Revalor" (20 mg estradiol-17beta and 140 mg TBA) with the highest concentrations found in liver and TBA could only be quantitated in fat.

It is firmly believed that sexual differentiation of the brain is linked with external genital differentiation in timing as an in utero event in the human. An extensive search for oestrogen, androgen and progestin receptors failed to show their presence despite adequate controls in cytosols from human fetal brain of gestational ages 14-20 weeks. It is possible that the receptors are present in levels so low that they are undetectable by present-day methods. Our results would indicate that hormonally influenced in utero brain sexual differentiation is most unlikely to occur as a mid-trimester event.

During the course of another investigation, three dogs had been castrated 3 months previously. Upon completion of the experiment, it was discovered that one dog presented a spontaneous prostatic adenocarcinoma of intraalveolar proliferative type at histology. Prostate weight of this dog before castration was estimated to be 22 g by tridimensional measurement at laparotomy and remained relatively constant (19 g) 3 months after castration. These results indicate that if regression had occurred in some cell populations (androgen-dependent) it was only partial and masked by growth of androgen-independent cells. Analysis of 12 individual steroids in peripheral blood and in prostatic tissue attested of a normal adrenal secretory activity. A series of 15 hydrolytic enzymes along with receptors for androgen, estrogen, and progesterone, were determined in prostatic tissue obtained at sacrifice. Enzymatic activities were those of typical epithelial cells, and most of them remained relatively high despite low levels of circulating testosterone. However, two markers of androgen action in dog prostate, acid phosphatase and arginine esterase, were significantly reduced. Receptor levels were similar to those of castrated animals. Thus, cancer cells had probably retained some androgen sensitivity.