Sorption and desorption of seven steroidal synthetic progestins in five agricultural soil-water systems.

Manure fertilization and wastewater irrigation can introduce the biologically potent synthetic progestins into agricultural soils, causing endocrine disruption in organisms of nearby surface waters. Therefore, this study investigated the sorption and desorption potential of etonogestrel, medroxyprogesterone, gestodene, norgestrel, cyproterone acetate, levonorgestrel, and dienogest in five agricultural soil-water systems. Sorption data were well-described by the linear sorption model. In most batch systems, cyproterone acetate exhibited the highest affinities for soils, followed by etonogestrel, medroxyprogesterone, levonorgestrel, gestodene, norgestrel, and dienogest. The sorption magnitudes (logKoc or logKd) were significantly correlated with the progestin hydrophobicities (R2 = 0.72-0.86, p < 0.05). The Kd values of the progestins were also significantly correlated with organic carbon content and pore volumes of the soils (R2 = 0.68-0.98, p < 0.05). In addition, 0.5 M urea resulted in 3-19% decreases in Kd values of the progestins. Taken together, these data indicated that hydrophobic partitioning interaction, hydrogen bonding interaction, and pore filling were the sorption mechanisms for the progestins in soil-water systems. No significant desorption hysteresis was observed for the progestins, indicating that they can be readily desorbed under rainfall or irrigation events. Based on the sorption and desorption data, we estimated the dynamic transport of the progestins in conventional agricultural management systems, and predicted the concentrations of the progestins as a function of soil-sorbed concentration, water-soil ratio, and dilution factor of receiving waters. This study will improve the understanding of the risks posed by the progestins under field-scale hydrological conditions.

Development of an LC-MS/MS method to quantify sex hormones in bovine milk and influence of pregnancy in their levels.

Hormones work in harmony in the body, and this status must be maintained to avoid metabolic disequilibrium and the subsequent illness. Besides, it has been reported that exogenous steroids (presence in the environment and food products) influence the development of several important illnesses in humans. Endogenous steroid hormones in food of animal origin are unavoidable as they occur naturally in these products. The presence of hormones in food has been connected with several human health problems. Bovine milk contains considerable quantities of hormones and it is of particular concern. A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method, based on hydroxylamine derivatisation, has been developed and validated for the quantification of six sex hormones in milk [pregnenolone (P5), progesterone (P4), estrone (E1), testosterone (T), androstenedione (A) and dehydroepiandrosterone (DHEA)]. This method has been applied to real raw milk samples and the existence of differences between milk from pregnant and non-pregnant cows has been statistically confirmed. Basing on a revision of existing published data, it could be concluded that maximum daily intakes for hormones are not reached through milk ingestion. Although dairy products are an important source of hormones, other products of animal origin must be considered as well for intake calculations.

[Simultaneous determination of 7 female sex hormones in essential oil by high performance liquid chromatography-tandem mass spectrometry with isotope dilution].

A reliable ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for the simultaneous determination of 7 female sex hormones (estriol, estradiol, estrone, ethinyloestradiol, dienestrol, hexestrol, diethylstilbestrol) in essential oil was developed. The sample was extracted by ethylacetate-normal hexane solution (2:98, v/v) and the extract was purified by a silica solid phase extraction-based clean-up column. Then, the analytes were separated on an ACQUITY UPLC BEH SHELD RP18 column (100 mm x 2.1 mm, 1.7 microm) in gradient elution with the mobile phases of water and acetonitrile. The separated compounds were detected with a Waters Xevo TQ MS tandem quadrupole mass spectrometer operated in negative electro-spray ionization using multiple reaction monitoring mode. Estriol-D3, estradiol-D3 and diethylstilbestrol-D6 were used as the internal standards to reduce the matrix effects. The limits of detection and quantitation for the 7 female sex hormones in essential oil were 0.3 -7 microg/kg and 1-20 microg/kg, respectively. Good linear relationships and high correlation coefficients (r2 > or = 0.997) were obtained in the mass concentration range of 20-500 microg/L. The average recoveries were 88.5%-114.8% and the intra-assay relative standard deviations were 4.8%-18.9% at the spiked levels of 20-500 microg/kg. Finally, a total of 12 samples randomly collected from different supermarkets in Zhejiang Province were screened for the 7 female sex hormones by the proposed method. The results showed that only one sample contained estradiol and estrone.

Fecal steroid monitoring for assessing gonadal and adrenal activity in the golden eagle and peregrine falcon.

We examined the efficacy of noninvasive monitoring of endocrine function via fecal steroid immunoassays in the golden eagle and peregrine falcon. High-pressure liquid chromatography analyses of fecal glucocorticoid metabolites (fGCM) revealed that minor percentages of immunoreactive fGCM co-eluted with [(3)H]corticosterone in both sexes of the eagle (2.5-2.7%) and falcon (7.5-11.9%). In contrast, most fecal estrogen metabolites in eagle and falcon females co-eluted with radiolabeled estradiol-17beta ([(3)H]; 57.6, 64.6%, respectively) or estrone ([(3)H]; 26.9, 4.1%, respectively). Most fecal progestin metabolite immunoreactivity in the female eagle (24.8%) and falcon (21.7%) co-eluted with progesterone ([(14)C]). Most fecal androgen metabolite immunoreactivity in eagle (55.8%) and falcon (63.7%) males co-eluted with testosterone ([(14)C]). Exogenous adrenocorticotropin hormone induced increased fGCM excretion above pre-treatment in both species, but only significantly (P < 0.05) in the eagle. Both species showed increased fGCM after saline administration, suggesting the detection of 'handling stress.' Both species exhibited enterohepatic and renal recirculation of administered steroids as demonstrated by biphasic and triphasic excretion patterns. Thus, noninvasive fecal hormone monitoring is a valid and promising tool for assessing gonadal and adrenal status in rare and threatened birds-of-prey.

Assessment of total effective xenoestrogen burden in adipose tissue and identification of chemicals responsible for the combined estrogenic effect.

Test systems to screen for estrogenicity and appropriate biomarkers of human exposure are required for epidemiological studies of endocrine disruption. We addressed these issues by developing and standardising a method to assess the total estrogenic xenobiotic burden in human adipose tissue. In this study, which is the continuation of a previous work, we have improved the protocol for extensive fractionation of a higher number of tissue samples in order to investigate bioaccumulated xenoestrogens that are candidates for estrogenicity and to assess their combined estrogenic effect. This was achieved by extensive HPLC separation of xenoestrogens from endogenous hormones followed by testing of individual fractions in the E-Screen test for estrogenicity. Organochlorine pesticides, PCBs and halogenated bisphenols and alkylphenols were collected in the most lipophilic fractions, followed by progestins, androgens and estradiol esters, and then by steroidal estrogens; phyto- and myco-estrogens were collected around the end of the run. These results were confirmed by exhaustive chemical analysis. In 458 human adipose tissue samples, the total effective xenoestrogen burden was positive in 75% of samples in the pooled fraction that contained organohalogenated xenoestrogens (mean 515.3 pM Eeq/g lipid; range 0-14.5 nM) and in 82% of samples in the pooled fraction where natural estrogens eluted (mean 696.6 pM Eeq/g lipid; range 0-12.9 nM). Organochlorine pesticides emerged as candidate chemicals for the estrogenicity of the first pooled fraction, because DDT and derivatives were present in 98.3% of the samples. However, no correlation was found between the concentration of any single chemical and the estrogenicity determined in the bioassay. There may be several reasons for this lack of concordance: (i) the estrogenic effects depicted in the E-Screen bioassay are a consequence of the combined effect of several organohalogens or (ii) the proliferative effect is due to other chemicals not measured. Because additive, synergistic or antagonistic mechanisms may account for the final effect observed in the pooled fractions, the approach proposed in this work is more appropriate for exposure assessment in epidemiological studies than the determination of individual chemicals in human samples.

CD spectrometric methods for the simultaneous determination of ethisterone and its delta5-isomer.

Quick and accurate direct and indirect circular dichroism (CD) spectrometric methods were developed for the simultaneous determination of ethisterone (17alpha-ethinyl-17-hydroxy-4-androstene-3-one) and its delta(5)-isomer (delta(5)-ethisterone). The direct method is based on the selective negative Cotton effect of the delta(4)-3-oxo group in ethisterone (negative maximum at 348 nm in dioxan) and measurement of the ellipticity at 296 nm (positive maximum of delta(5)-ethisterone), where the measured ellipticity is the sum of those of the two isomers. In the indirect procedure delta(5)-ethisterone is transformed to ethisterone by base-catalysed isomerization and the ellipticities are measured at 339 nm in ethanol before and after isomerization. Preliminary experiments show the usefulness of CD detector in the HPLC determination of the mixture of the isomers. A major advantage of the direct CD spectrometric and the HPLC/CD methods is that the delta(5)-isomer with extremely low UV activity can also be directly measured with high sensitivity.

Determination of steroid hormones in oral contraceptives by high-performance liquid chromatography.

The aim of this research was to standardize a high-performance liquid chromatographic method for quantitative determination of steroid hormones, like ethinylestradiol (ETE), levonorgestrel (LEVO), and gestodene (GEST), in commercially available oral contraceptives (OCs). The combination ETE-LEVO was analyzed using a LiChrospher 100 RP-8 column (5 microns, 125 x 4 mm) in LiChroCART, with a mobile phase constituted of acetonitrile: water (60:40 v/v). Using the same column, ETE-GEST was analyzed with a mobile phase constituted of acetonitrile:water (50:50 v/v) at pH 7.5 adjusted with 0.02 M ammonium hydroxide. For both methods, a flow rate of 0.8 mL/min was utilized and detection was carried out at 215 nm. All analyses were performed at room temperature (24 +/- 2 degrees C). Calibration curves for ETE-LEVO were obtained using solutions with concentration ranges from 2.40 to 60.0 micrograms/mL (ETE), and from 12.0 to 300.0 micrograms/mL (LEVO). Calibration curves for ETE-GEST were obtained using solutions with concentration ranges from 2.40 to 60.0 micrograms/mL (ETE), and from 9.0 to 160.0 micrograms/mL (GEST). Correlation coefficients obtained were from 0.9999 to 0.9990. Coefficients of variation for samples containing ETE-LEVO were 0.47% and 0.38%, respectively. For samples with ETE-GEST they were 0.39% and 0.44%, respectively. The average recovery for samples with ETE-LEVO was 103.46% and 100.78%, respectively. For samples containing ETE-GEST it was 100.89% and 101.03%, respectively.

A sensitive enzyme immunoassay (EIA) for the determination of melengestrol acetate (MGA) in adipose and muscle tissues.

The development of a sensitive screening method of MGA residues in bovine perirenal fat and muscle based on a competitive microtitration plate enzyme immunoassay is described. The samples were extracted with petroleum ether and purified with octadecyl-silica-cartridges. The detection limit for fat was 0.4 ng/g andfor muscle tissue 0.05 ng/g, much lower than requiredfor reliable detection of positive samples. The mean recovery rates of fortified samples amount to 75%, the mean intraassay variations to 7% and the interassay variation to 13%. Determination limits were validated for fat at 2 ng/g and for muscle at 0.1 ng/g. The efficiency of the new screening method was successfully demonstrated by the direct comparison to GC-MS and LC-MS methods performed at natural positive samples originating from an animal experiment in which the labelled dose (0.5 mg per animal and day) with and without a 48 h withdrawal period or 3-fold or 10-fold the amount of MGA, respectively, was fed to Holstein Frisian heifers. In conclusion, this new screening method can be used for sensitive determination of MGA residues in adipose tissues even after low treatment doses or longer withdrawal periods.

Elevated serum prolactin level with high-dose estrogen contraceptive pills.

BACKGROUND AND OBJECTIVES: There are currently groups of women using high-dose estrogen contraceptive pills, especially in the developing countries. The aim of this study was to determine the correlation between the duration of contraceptive pill intake, the dose of steroid contained in the contraceptive pills and the incidence and degree of serum prolactin level elevation in those women. STUDY DESIGN: This study was conducted in 100 contraceptive pill users. Women were randomly selected for this study with an age range from 19 to 35 years and duration of contraceptive pill intake from 6 to 120 months. Cases were classified into two groups. The first group (50 cases) were taking high-dose estrogen pills (50 micrograms) and the second group (50 cases) were taking low-dose estrogen pills (30 micrograms). RESULTS: The results of the present study showed that there was a significant elevation in serum prolactin level in both groups, with a more significant elevation in the high-dose pill users. CONCLUSIONS: There is a positive relationship between serum prolactin level and the duration of pill intake and their steroid content, and this relationship is not related to the age and parity of the women. The groups of women studied are scheduled for follow-up to determine if there is any future drawback which results as a consequence of the developed hyperprolactinemia. Prolactin determination should be considered for all women prior to pill intake. This determination of serum prolactin level prior to pill use will be useful in the evaluation of the future relationship between the estrogen content of the pills and the later development of hyperprolactinemia.

Binding activity of 5alpha-reduced gestagens to the progestin receptor from African elephant (Loxodonta africana).

Recent findings in the African elephant (Loxodonta africana) indicate that the major progestins contained within and biosynthesized by corpora lutea are 5alpha-reduced metabolites and that progesterone is quantitatively of minor importance. The specific gestagenic action within the reproductive tract of elephants was determined by measurement of relative binding affinity of the respective progestins to the gestagen receptor extracted from elephant endometrium. The cytosol was incubated with 40 nmol/liter [3H]ORG-2058 and increasing concentrations of the tested progestin. Progesterone (P4), 5alpha-pregnane-3,20-dione (DHP), and 5alpha-pregnane-3alpha-ol-20-one (5alpha-P-3OH) were used. The competition for binding sites on the progestin receptor was shown by decreasing counts measured after extraction with scintillation fluid. The progestin concentration which induced a 50% reduction of measured counts was estimated (C50) and relative binding affinity of progesterone to other progestins was calculated (RBA = C50progestin/C50p4). The relative binding affinity of DHP to P4 at the gestagen receptor of elephant endometrium was equivalent. The other 5alpha-reduced progestin (5alpha-P-3OH) showed no competition to the [3H]ORG-2058 receptor binding. We conclude that the biological significance of P4 and DHP at the receptor level is very similar. The higher quantitative levels of DHP in corpus luteum and serum support the hypothesis that this progestin is the major gestagen in the elephants, whereas 5alpha-P-3OH is an inactive metabolite.

Simultaneous determination of ethinylestradiol and levonorgestrel in oral contraceptives by derivative spectrophotometry.

A method for determining ethinylestradiol (ETE) and levonorgestrel (LEV) in mixtures by first-derivative spectrophotometry is described. The procedure does not require any separation step. Measurements are made at the zero-crossing wavelengths and the calibration graphs are linear up to 26 and 33 micrograms ml-1 of ETE and LEV, respectively. The method was applied to the determination of both compounds in five different Spanish commercial low-dose oral contraceptives. Similar results were obtained by an HPLC method.

Pharmacodynamic effects of depot-medroxyprogesterone acetate (DMPA) administered to lactating women on their male infants.

Normal postpartum women, who had a spontaneous vaginal delivery of one full-term male infant, free of congenital abnormalities and other diseases, were recruited for this study. Thirteen women received 150 mg depot-medroxy-progesterone acetate (DMPA), intramuscularly on days 42 + 1 and 126 + 1 postpartum. Infants of nine mothers, who did not receive DMPA, served as controls. Blood samples were collected from treated mothers on days 44, 47, 74, 124, 128, and 130 postpartum for medroxyprogesterone acetate (MPA) measurements. Four-hour urine collections were obtained from all 22 infants in the morning on days 38, 40, 42, 44, 46, 53, 60, 67, 74, 88, 102, 116, 122, 124, 126, 128, 130, and 137. Urinary follicle stimulating hormone (FSH), luteinizing hormone (LH), unconjugated testosterone, and unconjugated cortisol were measured by radioimmunoassay, and serum MPA and urinary MPA metabolites were measured by gas chromatography-mass spectrometry (GC-MS). No MPA metabolites could be detected in the urine of the infants from the DMPA-receiving mothers. Hormonal profiles in the urine samples were not suppressed in comparison with those of the control infants. The present study demonstrates that DMPA, administered to the mother, does not influence the hormonal regulation of the breast-fed normal male infant.

Sexual differentiation of the human midtrimester brain.

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.

[Radioimmunologic determination of the progestagen dienogest in plasma and saliva]. / Radioimmunologische Bestimmung des Progestagens Dienogest im Plasma und im Speichel.

Following oral administration of 2 mg of dienogest (17 alpha-cyanomethyl-17 beta-hydroxy-4,9-estradien-3-one) to female volunteers, the dienogest concentration courses in plasma and saliva were determined by means of a specific radioimmunoassay (RIA). Three different procedures of the plasma sample preparation prior to the RIA were compared. The dienogest RIA was directly applied to saliva. There is a high correlation between the dienogest concentrations in plasma and saliva. The dienogest plasma elimination half life of about 9 hrs is not significantly different from that derived from saliva. The salivary dienogest concentrations indicate a relatively high non-protein bound portion of this steroid drug in plasma. Following repeated oral administration of dienogest (tau = 24 hrs), there is no significant cumulation of plasma dienogest.

[Bovine serum albumin conjugates of the new progestagen dienogest, synthesis and immunogenic properties]. / Rinderserumalbuminkonjugate des neuen Progestagens Dienogest--Syntheseund immunogene Eigenschaften.

In order to develop a radioimmunoassay for the new progestagen dienogest (STS 557, 17 alpha-cyanomethyl-17 beta-hydroxy-estra-4,9-dien-3-one), bovine serum albumin (BSA) conjugates of STS 557-3-carboxymethyloxime and of STS 557-11-hemisuccinate were synthesized as antigens for the production of antisera. It was proved that an excess of isobutylchlorocarbonate in the coupling reaction using the "mixed anhydride method" results in an acylation of free NH2-groups in the BSA. By the immunization of rabbits with the STS 557-antigen-antisera of high specificity and affinity to STS 557 were produced. Endogenous steroids show no cross reaction with the STS 557-antisera. Steroids with a 17 alpha-CH2CN-group, being obtained by chemical synthesis or microbial transformation, compete with STS 557 for the binding positions of the antibodies to a different extent.

A case-control study of breast cancer in relation to the use of steroid contraceptive agents.

In a case-control study of 141 cases of breast cancer and 279 control patients from the Royal Prince Alfred and Westmead Hospitals during 1980-1982, we found similar risk factors to those reported for other populations. There was no statistically significant evidence of an increased risk of cancer from the use of oral contraceptive agents; the crude estimate of relative risk for patients who had used oral contraceptive agents at some time was 1.3 with 95% confidence limits of 0.8 and 1.9. After adjustment for other risk factors (age at first live birth, age at menarche, number of pregnancies, menopausal status, bilateral oophorectomy and years of education), the estimate of the relative risk of ever having used an oral contraceptive agent was 0.9 with 95% confidence limits of 0.6 and 1.5. Further analysis in terms of duration of use and dosage also provided no evidence of an increased risk.

Localization of a synthetic progestin in the reproductive organs of the female baboon.

The uptake and retention of a radiolabeled synthetic progestin, ORG 2058, was studied in the female reproductive system of the baboon. Four estrogen-primed baboons were injected intravenously with 2.5 micrograms/kg body weight of 3H-ORG 2058. One animal, which served as a control, received an additional injection of 2.5 mg/kg body weight of unlabeled progesterone. One hour after the injections, the animals were killed and the uterus, cervix, oviduct, vagina, and labia were removed and processed for autoradiography. The cells in the germinative layers of the stratified squamous epithelium of the cervix, vagina, and labia demonstrated nuclear localization of the label. The columnar epithelium, both surface and glandular, of the uterus and cervix sequestered the synthetic steroid; however, the nuclei of the epithelium lining the oviduct were unlabeled. The nuclei of the fibroblasts and of the smooth muscle cells were labeled in all the organs studied. These preliminary observations suggest that there is a stage in the reproductive cycle in which progesterone receptors are contained in the stromal cells of the oviduct but are absent in the epithelium.

Oestrogen receptor assay. False positive analysis?

The influence of unlabelled oestradiol, DES, testosterone and R-5020/org 2058 on tritiated oestradiol binding was investigated in 162 ER positive cases of patients with primary breast carcinoma. A dextran-coated charcoal as well as a sucrose gradient method was applied. In 122 cases only unlabelled oestradiol and DES significantly displaced the binding of labelled oestradiol. In the remaining 40 cases, oestradiol, DES, as well as testosterone and R-5020/org 2058 were able to displace the high-affinity, saturable binding of tritiated oestradiol equally. Possible explanations of this new discovery are discussed.