A survey of 17α-ethinylestradiol and mestranol residues in Hawkesbury River, Australia, using a highly specific enzyme-linked immunosorbent assay (ELISA) demonstrates the levels of potential biological significance.

This study reports on the potential status of 17α-ethinylestradiol (EE2) and mestranol (MeEE2) residues in aquatic environments in New South Wales (NSW), Australia, based on the analysis by a specific ELISA we developed. Polyclonal antibodies were raised against the EE2 hapten with a linker attached at the C3-position to direct the antibody binding towards the ring D of EE2/MeEE2. Using this approach, an ELISA highly specific to EE2 and MeEE2 was successfully developed, showing less than 3.1% cross-reactivity (% CR) with other major steroidal sex hormones and their derivatives. The assay performed with the limit of detection (LOD) of 0.04 ± 0.01µg/L for both EE2 and MeEE2, and the limit of quantitation (LOQ) of 0.05 ± 0.01ng/L when it was coupled with the SM2-Biobeads solid phase extraction. Prior to conducting the survey study, it was validated against the gas chromatography-mass spectrophotometry (GC-MS) method, which showed high correlation with R2 of 0.934. Fresh surface water samples collected at different sites along Hawkesbury River in New South Wales (NSW) were analyzed for the EE2/ MeEE2 residues using the developed ELISA. The EE2/MeEE2 levels were found to range between 4.1 and 8.3ng/L in Emigrant Creek, NSW, where the primary activity was macadamia plantation, and higher levels between 15 and 29ng/L in South Creek, NSW, Greater Western Sydney at sites upstream and downstream of the municipal sewage treatment plants.

17α-Ethynylestradiol alters the peritoneal immune response of gilthead seabream.

17α-Ethynylestradiol (EE2), a synthetic estrogen used in most oral contraceptives pills and hormone replacement therapies, is found in many water bodies, where it can modulate the fish immune response. EE2 acts as an endocrine disruptor in gilthead seabream, Sparus aurata L., a marine teleost fish of great economic value in Mediterranean aquaculture, as it induces hepatic vitellogenin gene (vtg) expression. Moreover, EE2 also alters the capacity of gilthead seabream to appropriately respond to infection although it does not behave as an immunosuppressor. Nevertheless, these previous studies have mainly focused on the head kidney leukocytes and no information exists on peritoneal leukocytes, including mast cells. In the present work, juvenile gilthead seabream fish were fed a pellet diet supplemented with EE2 for 76 days and intraperitoneally injected with hemocyanin plus imject alum adjuvant at the end of EE2 treatment and 92 days later, and the peritoneal immune response was analyzed. EE2 supplementation induced vtg expression but returned to basal levels by 3 months post-treatment. Interestingly, gilthead seabream peritoneal leukocytes express the genes encoding for the nuclear estrogen receptor α and the G protein-coupled estrogen receptor 1 and the dietary intake of EE2 induced these expression. Moreover, EE2 induced an inflammatory response in the peritoneal cavity in unvaccinated fish, which was largely maintained for several months after the cessation of the treatment. However, the impact of EE2 in vaccinated fish was rather minor and transient. Taken together, the study provides fresh information about endocrine immune disruption, focusing on peritoneal leukocytes.

Electrochemical immunosensor for ethinylestradiol using diazonium salt grafting onto silver nanoparticles-silica-graphene oxide hybrids.

This work describes the preparation of an electrochemical immunosensor for ethinylestradiol (EE2) based on grafting of diazonium salt of 4-aminobenzoic acid onto a glassy carbon electrode modified with silver nanoparticles/SiO2/graphene oxide hybrid followed by covalent binding of anti-ethinylestradiol (anti-EE2) to activated carboxyl groups. A competitive immunoassay was developed for the determination of the hormone using peroxidase-labeled ethinylestradiol (HRP-EE2) and measurement of the amperometric response at -200mV in the presence of hydroquinone (HQ) as redox mediator. The calibration curve for EE2 exhibited a linear range between 0.1 and 50ng/mL (r(2)=0.996), with a detection limit of 65pg/mL. Interference studies with other hormones related with EE2 revealed the practical specificity of the developed method for the analyte. A good reproducibility, with RSD=4.5% (n=10) was also observed. The operating stability of a single bioelectrode modified with anti-EE2 was maintained at least for 15 days when it was stored at 4°C under humid conditions between measurements. The developed immunosensor was applied to the analysis of spiked urine with good results.

The effect of 17α-ethynylestradiol on steroidogenesis and gonadal cytokine gene expression is related to the reproductive stage in marine hermaphrodite fish.

Pollutants have been reported to disrupt the endocrine system of marine animals, which may be exposed through contaminated seawater or through the food chain. Although 17α-ethynylestradiol (EE2), a drug used in hormone therapies, is widely present in the aquatic environment, current knowledge on the sensitivity of marine fish to estrogenic pollutants is limited. We report the effect of the dietary intake of 5 µg EE2/g food on different processes of testicular physiology, ranging from steroidogenesis to pathogen recognition, at both pre-spermatogenesis (pre-SG) and spermatogenesis (SG) reproductive stages, of gilthead seabream (Sparus aurata L.), a marine hermaphrodite teleost. A differential effect between pre-SG and SG specimens was detected in the sex steroid serum levels and in the expression profile of some steroidogenic-relevant molecules, vitellogenin, double sex- and mab3-related transcription factor 1 and some hormone receptors. Interestingly, EE2 modified the expression pattern of some immune molecules involved in testicular physiology. These differences probably reflect a developmental adjustment of the sensitivity to EE2 in the gilthead seabream gonad.

Immunosensors for estradiol and ethinylestradiol based on new synthetic estrogen derivatives: application to wastewater analysis.

Novel electrochemical immunosensors for sensitive detection of 17-ß estradiol (E2) and ethinylestradiol (EE2) are described on the basis of the use of magnetic beads (MBs) as solid support and screen-printed electrodes as sensing platforms. Four synthetic estrogen derivatives containing either a carboxylic group or an amine group at the C-3 position were synthesized and covalently bound to MBs functionalized with amine or carboxyl groups, respectively. The assay was based on competition between the free and immobilized estrogen for the binding sites of the primary antibody, with subsequent revelation using alkaline phosphatase-labeled secondary antibody. Preliminary colorimetric tests were performed in order to validate the applicability of the synthetic estrogens to immuno-recognition and to optimize different experimental parameters. In a second step, electrochemical detection was carried out by square wave voltammetry (SWV). Under the optimized working conditions, the electrochemical immunosensors showed a highly sensitive response to E2 and EE2, with respective detection limits of 1 and 10 ng/L. Cross-reactivity evaluated against other hormones demonstrated an excellent selectivity. The developed devices were successfully applied to analysis of spiked and natural water samples. These new immunosensors offer the advantages of being highly sensitive, easy, and rapid to prepare, with a short assay time.

Natural and synthetic estrogens modulate the inflammatory response in the gilthead seabream (Sparus aurata L.) through the activation of endothelial cells.

Sex steroids are known to deeply alter processes other than fish reproduction, including fish growth, intermediary metabolism, osmoregulation and immunity. We have previously reported that 17ß-estradiol (E(2)), the main fish estrogen, promotes the mobilization of acidophilic granulocytes from the head kidney, the bone marrow equivalent in fish, to the gonad in the bony fish gilthead seabream (Sparus aurata L.). The aim of this study was to investigate the effects of E(2) and 17α-ethinylestradiol (EE(2)), an endocrine disruptor with strong estrogenic effects commonly found in the aquatic environment, on the ability of gilthead seabream endothelial cells (ECs) to promote leukocyte infiltration. E(2) and EE(2) were seen to affect ECs in different ways. Thus, E(2) was able to increase the production of nitric oxide (NO) and up-regulate the expression of the key activation markers, interleukin-1ß, CC chemokine ligand 4, interleukin-8, E-selectin and matrix metalloproteinase 9, when used alone or combined with bacterial DNA. In contrast, EE(2) failed to affect NO release and reduced the up-regulation of the above genes promoted by bacterial DNA. Moreover, we found that leukocyte adhesion to ECs was enhanced by E(2) treatment. Collectively, these results suggest that estrogens modulate fish leukocyte trafficking during an inflammatory process by activating ECs.

Field study using two immunoassays for the determination of estradiol and ethinylestradiol in the aquatic environment.

The effluent of four sewage treatment plants (STP) and eight surface water samples from the river Rhine in Germany and two smaller rivers were monitored for the hormones estradiol (E2) and ethinylestradiol (EE2). The studied STPs are using different treatment processes. Two facilities include an activated sludge treatment, one is a constructed wetland, and one is just an aerated lagoon. For analysis of E2 and EE2 in the aquatic environment two immunoassays have been developed allowing a very cost-effective screening for both hormones in environmental samples. Detection limits could be established at 0.05 ng L(-1) for E2 and 0.01 ng L(-1) for EE2, taking a 50-fold enrichment into account. Median concentrations for E2 and EE2 in effluent samples were 12 and 1.8 ng L(-1), in surface water 4.0 and 0.7 ng L(-1), respectively. The highest estrogen concentrations were found in the effluent of the lagoon, equipped with very basic means of wastewater treatment.

Characterisation of antibodies and analytes by surface plasmon resonance for the optimisation of a competitive immunoassay based on energy transfer.

The determination of binding constants using surface plasmon resonance (SPR) was introduced to optimise a competitive homogeneous fluorescence energy-transfer immunoassay (ETIA) before labelling. Steroids were chosen as model for the detection of three analytes estrone, estradiol and ethinylestradiol--by taking three polyclonal antibodies (anti estrone-, anti estradiol- and anti estrogen-antibodies) and the corresponding analyte derivatives used for the immunisation. The active concentration of the antibodies was determined before and after labelling. Inhibition curves were recorded using SPR for all possible combinations of analyte, antibody, and analyte derivatives. The experiments revealed that the active antibody concentration can be reduced to 30% whereas the antibody affinity is not affected by the labelling process. Limits of the use of SPR for determination of affinity constants in solution are discussed. All possible ETIA calibration for the quantification of estrone and estradiol was performed. The lower limits of detection for estrone (0.06 microg L(-1)) and estradiol (0.17 microg L(-1)) were reached with the anti-estrogen IgG and its derivative

Absence of antisteroid antibodies in oral contraceptive users presenting with vascular events.

Previous reports speculated that vascular events could be related to the development of antibodies against synthetic steroids contained in oral contraceptives or other hormonal treatments. This study describes original immunoassays designed to detect antisynthetic steroid antibodies. In a first step, the assays were characterized and validated using animal-raised antisteroid antibodies. In a second step, a population of 88 oral contraceptive users, 47 of them having developed a vascular thrombosis during synthetic steroid use and 41 serving as healthy control users, were tested. Detection of antibodies against ethinylestradiol, levonorgestrel, norethisterone, cyproterone acetate, and gestodene showed that the values obtained in normal oral contraceptive users as well as thrombosis patients are very low, and show no statistically significant difference between the two groups tested. Taken together, these data indicate that the "immunological hypothesis" related to antisteroid antibodies is unlikely to explain the pathogenesis of vascular events in oral contraceptive users.

Antisteroid immune complexes and vascular thrombosis during steroid hormone therapy.

To test an immunological hypothesis proposed to explain the pathogenesis of cerebrovascular thrombosis in steroid users, circulating immune complexes were assayed in the sera from 6 control subjects, 14 ever users of oral contraceptive having developed a neurological ischaemic accident, and 7 patients with the same clinical history during use of other sex steroid not containing ethinylestradiol. Beaumont's ammonium sulfate and polyethylene glycol precipitation methods, together with a specific method of isolation of circulating immune complexes using affinity chromatography on Protein A, were used. Radioactivity from labeled ethinylestradiol added to the sera before precipitation was monitored in the precipitates to detect anti-ethinylestradiol antibodies. There were no significant differences for these parameters in the three groups. However, protein content and 3H-EE activity in the precipitates were equally and dramatically reduced after affinity chromatography in the three groups. These latter results do not support the presence of antibodies against ethinylestradiol in steroid users with cerebrovascular thrombosis. Moreover, our data suggest a lack of specificity of Beaumont's method for the isolation of immune complexes containing anti-ethinylestradiol antibodies.

Suppression of acute experimental allergic encephalomyelitis by the synthetic sex hormone 17-alpha-ethinylestradiol: an immunological study in the Lewis rat.

Induction of experimental allergic encephalomyelitis (EAE) in female Lewis rats led to the well-known clinical symptoms and histological signs. Treatment with the synthetic estrogen 17-alpha-ethinylestradiol (EE) from day -4 before induction until day 21 after induction resulted in partial suppression of these signs and symptoms. Analysis of the peripheral blood leukocyte (sub)populations in these treated animals indicated some remarkable changes. However, these changes were also observed without EE treatment. EE treatment of EAE rats resulted in a significant decrease of the relative weights of both thymus and spleen, which changes however were not reflected in the peripheral blood. Apparently the effects of EE treatment on EAE in the present experiments indicate an action locally at the site of the EAE lesion and do not seem to be mediated by gross changes in the levels of peripheral blood leukocytes.

Inflammatory reaction in endometriotic tissue: an immunohistochemical study.

A panel of monoclonal antibodies specific for macrophage subtypes appearing in early (27E10), down-regulatory (RM3/1) and late (25F9) stages of inflammation had been applied to 20 endometriotic implants of 14 women. Of those patients 9 were in the follicular phase of the cycle, two on danazol, one on LHRH-analogue (buserelin) and another two on oral contraceptives. Beside the macrophage subsets, antibodies against T4, T8 lymphocytes as well as proliferating cells (EN7/44 and Ki67) were examined. In all specimens immunologically competent cells could be detected to a varying degree and within the same implant different stages of inflammation were discernible. Endometriosis presented with signs of early inflammation indicated by 27E10+ macrophages and CD4+ lymphocytes (15 specimens) and with down-regulatory, late inflammatory reactions as shown by RM3/1+, 25F9+ macrophages and CD8+ lymphocytes (19 biopsies). Additionally, in 14 specimens cells of the EN7/44+ and Ki67+ type was detected. These preliminary results showed no significant correlation to either extension of endometriotic implants or adhesions or concomitant therapy and clearly indicate, that there is an immunological dynamic process within the lesion itself in addition to that one of the peritoneal fluid.

[Splenic artery thrombosis with Adepal. Pathogenic role of anti-ethinylestradiol antibody?]. / Thrombose de l'artère splénique sous Adépal. Rôle pathogène d'anticorps anti-éthinylestradiol?

We report a case of splenic artery thrombosis developed in a 54-year old woman after prolonged use of oral contraceptives. The diagnosis of the disease, difficult on clinical grounds, was confirmed by computerized tomography and selective arteriography. The presence of anti-ethinyloestradiol antibodies in the serum is suspected to be a risk factor for thrombosis associated with oral contraception.

Hyperhomocyst(e)inemia, anti-estrogen antibodies and other risk factors for thrombosis in women on oral contraceptives.

Hyperhomocyst(e)inemia was shown to be associated with vascular occlusion in atherosclerotic patients. We have conducted a study to determine if hyperhomocyst(e)inemia was also related to the vascular events observed in women on oral contraceptives, presumably having little or no atherosclerosis. Two hundred women receiving oral contraceptives were included in the study: 100 were healthy controls and 100 had documented vascular occlusion. Determination of serum homocyst(e)ine and anti-estrogen antibody levels wore performed under blind conditions. They were evaluated in logistic regression models in which age and smoking were also included. Women with vascular occlusion had higher levels of homocyst(e)ine (P less than 0.001) and of anti-estrogen antibodies (P less than 0.001) when compared to controls. They were also older (P less than 0.001) and more frequently smokers (P less than 0.05). The above mentioned variables were, in isolation, independent predictors of vascular occlusion. Moreover, a model assessing those variables and their interactions indicated that the levels of anti-estrogen antibodies and smoking increased the predictability in older women, as well as the levels of age-adjusted homocyst(e)ine. The study suggests that the above factors can identify women at risk and that determination of anti-estrogen antibodies and homocyst(e)ine levels may help to detect women predisposed to vascular occlusions when taking oral contraceptives.

PIP: This study was conducted to test the hypothesis that hyperhomocyst(e)inemia may be an additional risk factor for vascular occlusions in women taking oral contraceptives (OCs). A total of 200 women who were regular users of OC tablets containing 30 or 50 mcg ethinyl estradiol were studied: 100 were controls and 100 had documented vascular occlusion. Serum levels of homocyst(e)ine and anti-estrogen antibody were determined under blind conditions. These were evaluated in logistic regression models in which age and smoking were also included. Women with vascular occlusion had higher levels of homocyst(e)ine and anti-estrogen antibodies when compared to controls. They were also older and frequent smokers. The variables investigated--namely, anti-estrogen antibody, age-adjusted log, transformed homocyst(e)ine, age, and smoking--were independent predictors of vascular occlusions when considered in isolation. Moreover, a model assessing these variables and their interactions, indicated that the levels of anti-estrogen antibodies and smoking increased the predictability in older women, as well as the levels of age-adjusted log and homocyst(e)ine. The study suggests that these variables can identify women at risk, and the determination of anti-estrogen antibody and homocyst(e)ine levels may help to detect women predisposed to vascular occlusions when taking OCs.

The significance of circulating antiethinyl-estradiol antibodies (AEEA) in the occurrence of thrombosis in women while taking the pill.

The aim of the study was to investigate the hypothesis that oral contraceptives (OC) lead to antiethinyl-estradiol antibody (AEEA) synthesis which might in turn be responsible for the thrombo-embolic complications observed in women users. The trial included 428 women divided into a) 204 healthy female volunteers as the control group (CONT), b) 139 women who had suffered thrombo-embolic accidents (THR), c) 85 women suffering from recurrent fetal loss (RFL). In each of these three categories, 50% were pill users (OC+) and 50% were not (OC-). Specific immune markers of systemic autoimmunity and anticardiolipin antibodies were looked for, as they are thought to be present in increased amounts in thrombosis or recurrent fetal loss. The AEEA prevalence differed significantly (p less than 0.05) between the CONT OC+ (32%) and the CONT OC- (13%) women. It also differed (p less than 10(-5)) between the RFL OC+ (60%) and the RFL OC- (12%) women. It did not differ between THR OC+ (36%) and THR OC- (39%) women. Within the OC+ women, the difference between the THR and the CONT groups was not found to be significant, whereas it was found to differ significantly between the RFL and the CONT groups (odds-ratio RFL/CONT estimated at 3.20, confidence interval 1.53, 6.69). Within the OC-women, the AEEA prevalence was found to differ significantly between the THR (39%) and the CONT (13%) groups (odds-ratio THR/CONT estimated at 4.40, confidence interval 2.07, 9.38%) but not between the RFL and the CONT groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Oral contraceptives, sex steroid-induced antibodies and vascular thrombosis: results from 1318 cases.

The role of antiethinyl estradiol antibodies (anti EE Ab) and associated risk factors was evaluated in 1318 cases of venous or arterial thrombosis in oral contraceptives (OC) users, and compared to 61 non-users and 124 healthy current users. Anti EE Ab were absent in non-users and present in 33% of healthy users and 72% of those with thrombosis, either arterial or venous. Age, duration of use, hyperlipidaemia and smoking were factors associated with thrombosis only in women with an arterial disease. While the two predominant factors, anti EE Ab and smoking may be risk factors in their own right, the combination of both was found in 47.7% of women with thrombosis. It is proposed that thrombosis associated with OC use may be explained by an immunological disease in which anti EE Ab and their complexes with the circulating synthetic hormones may be harmful to the vessels, as also suggested by the type of lesions already described in OC users. The determination of anti EE Ab in healthy users may identify a group at risk of thrombosis.

PIP: The significance of antibodies against ethinyl estradiol (anti-EE-Ab) and other risk factors was discussed for a series of 1318 cases of venous and arterial thrombosis in oral contraceptive users, in comparison to 61 non-users and 124 health current pill users. The cases included 264 deep vein thromboses, 159 pulmonary embolism, 37 coronary artery, 33 systemic artery, 763 cerebrovascular artery thromboses, and 10 hepatic vein thromboses collected from 88 French hospitals from 1976-1988. There were 98 cases with successive or multiple sites involved. The mean age of contraceptive users with thrombosis was 32.1, compared to 28.8 in healthy users. Duration of use was slightly longer in affected users than healthy users, but some cases were affected as early as their 1st cycle. 87.2% had no related history. The anti-EE-Ab were absent in never users, averaged 318 c./min in pill users with thrombosis, but 60 in healthy pill users. There was no correlation between anti-EE-Ab level and dose or duration of pill use. Similar anti-EE-Ab levels were found in those with venous or arterial thrombosis, but women with arterial thrombosis were older, had used pills longer, had fewer predisposing factors of surgery or labor and delivery, but more frequent incidence of hyperlipidemia, smoking, and hypertension. The most frequent associated factors with thrombosis were presence of anti-ee-Ab and smoking: 15.6% smoked, 31.1% had anti-EE-Ab, and 47.6% had both, but only 9.5% had neither factor. It is interesting that lowering the estrogen dose of oral contraceptives has decreased the frequency of venous thrombosis, but not that of arterial thrombosis or mortality, nor anti-EE-Ab levels. The vascular lesions in arterial thrombosis seen in pill users are thought to resemble those in many autoimmune diseases.

Anti-estrogen antibodies in systemic lupus erythematosus: a quantitative evaluation of serum levels.

We measured the beta-estradiol binding capacity of serum gamma-globulins in four subject groups; 1) normal men, 2) normal women who had never taken oral contraceptives, 3) normal women who had a history of oral contraceptive use and, 4) patients with systemic lupus erythematosus (SLE). We used dextran-coated charcoal to strip endogenous estradiol from serum proteins, added 3H-estradiol, and measured its association with proteins in various electrophoretic fractions following zone separation on agarose gels. Most of the bound radioactivity was present in the albumin, beta and gamma-globulin fractions. Binding to gamma-globulins was elevated in SLE patients, and normal controls who had taken oral contraceptives, as opposed to other controls (p less than 0.005). Gamma-region radioactivity could be removed by protein-G adsorption prior to zone electrophoresis. Isoelectric focusing revealed a pattern of tritiated-E2 binding consistent with polyclonal B-cell activation in all groups. There was no correlation between the extent of gamma-region binding and the total serum immunoglobulin level for any of the groups studied, nor was there a correlation between E2 binding and anti-DNA titers in the SLE group. The average anti-estradiol antibody concentrations in SLE sera (assuming equimolar binding) was 105 ng/ml (95% CL = 92-118), whereas their average anti-DNA antibody concentration was in the microgram/ml range. Thus, quantitatively, the level of anti-estradiol antibodies is at least an order of magnitude lower than the anti-DNA antibodies characteristic of this disease.

[Myocardial infarction and anti-ethinylestradiol antibody. Apropos of a case in an 18-year-old woman]. / Infarctus du myocarde et anticorps anti-éthinylestradiol. A propos d'un cas chez une jeune femme de 18 ans.

An 18-year-old woman presented with a large anterior myocardial infarction. Her cardiovascular risk factors were cigarette smoking in moderation and oral contraception with a synthetic oestroprogestative pill prescribed a few months previously. Coronary angiography showed occlusion of the left anterior descending artery but no other lesions. Biological investigations excluded an abnormality of coagulation. Antibodies to synthetic steroids (ethinylestradiol and progesterone) and circulating immune complexes were found in the serum. The role of antiethinylestradiol antibodies in the mechanism of myocardial infarction is discussed. These antibodies are present in 30 per cent of women taking oral contraceptives and their titres are significantly higher in 90 per cent of women who develop vascular thrombosis unrelated to atherosclerosis. The mechanism of the thrombogenic action of the antibodies and circulating immune complexes is also considered.