Biotransformation of Ethinylestradiol by Whole Cells of Brazilian Marine-Derived Fungus Penicillium oxalicum CBMAI 1996.

In this study, we report our contribution to the application of whole cells of Brazilian marine-derived fungi in the biotransformation of ethinylestradiol 1. A preliminary screening with twelve marine-derived fungi strains revealed that the fungus Penicillium oxalicum CBMAI 1996 promoted the biotransformation of ethinylestradiol 1. Then, P. oxalicum CBMAI 1996 was employed in the reactions in decaplicate in order to purify and characterize the main biotransformation products of ethinylestradiol 1. Compounds 1b and 1c were characterized by NMR, HRMS, [α]D and mp. Compound 1b was also characterized by single crystal X-ray diffraction. In addition, kinetic monitoring of the biotransformation of ethinylestradiol 1 by P. oxalicum CBMAI 1996 was evaluated in this study in order to obtain high yields of compounds 1b and 1c with a reduction of the reaction time. In this work, we proposed a biotransformation pathway of ethinylestradiol 1, which suggests the presence of several enzymes such as phenol oxidases, monooxygenases, and ene-reductases in the fungus P. oxalicum CBMAI 1996. In summary, the rapid biodegradation of ethinylestradiol 1 and compounds 1b and 1c also has an environmental relevance, since ethinylestradiol 1 and other steroidal compounds are improperly discarded in the environment, and part of these compounds are displaced into the oceans.

Ethinylestradiol and its effects on the macrophages in the prostate of adult and senile gerbils.

Prenatal and neonatal exposure to estrogenic compounds, such as ethinylestradiol (EE), promotes a variety of developmental disorders, including malformations and alterations in the morphology of glands, such as the prostate gland. Therefore, the aim of this study was to evaluate the morphological effects of neonatal exposure to EE on prostatic tissue and on the identification and quantification of gerbil gland macrophages in adult and senile Mongolian gerbils. The animals were exposed to EE (10 µg/kg/day) and to the vehicle, mineral oil (100 µL) (control group) during the first 10 days of postnatal life (lactation period). Adult gerbils were euthanized at 120 days and senile gerbils at 12 months of age. Our findings permitted verification of the presence of areas with proliferative foci in the prostate glandular portions in the adult and senile animals exposed to EE. There was also an increase in macrophages in the prostate tissue of adult and senile gerbils; these cell types alter the stromal microenvironment and possibly modify the interactions between the epithelium and stroma. Neonatal exposure to EE changes the pattern of prostatic development, leading to alterations in the arrangement of cells, including macrophages, and may be related to the onset of proliferative disorders in the prostate of adult gerbils and during aging.

Biosorption of 17α-ethinylestradiol by yeast biomass from ethanol industry in the presence of estrone.

Yeast biomass from ethanol industry (YB) was evaluated as a biosorbent to 17α-ethinylestradiol (EE) alone and along with estrone (EST). This material is rich in sorption sites and has a good cost-benefit ratio, since it is an industrial residue largely produced (around 30 g for each liter of ethanol). A 2k-factorial design was carried out to evaluate the sorption capacity of YB for EE considering the variables pH, biosorbent dose (BD), and ionic strength (IS), at two hormone concentration (HC) levels. The best conditions assessed for individual EE adsorption (pH = 10, IS = 0.1 mol/L, and BD = 0.5 mg/L) were also established for adsorption carried out in the presence of EST. Individuals EE and EST experimental sorption capacities (SCexp) were, respectively, 24.50 ± 0.07 and 0.80 ± 0.07 mg/g, fairly similar to Qmax (EE, 21.41 ± 1.27 mg/g; EST, 0.93 ± 0.075 mg/g) from Langmuir model. The Freundlich model best fitted the experimental data for EE adsorption (r2 = 0.9925; χ2 = 0.5575). The study carried out in the presence of EST showed an associative/competitive sorption process between EE and EST, which may be explained by their similar chemical structures and organic carbon-water partition coefficients Koc.

Stability of immobilized laccase on Luffa Cylindrica fibers and assessment of synthetic hormone degradation.

In this work were studied the pH, thermal, and storage stability of free and immobilized laccases. Enzymes were produced by Pleurotus ostreatus on potato dextrose (PD) broth and potato dextrose modified (PDM) broth, and immobilized using Luffa cylindrica fibers as support. Both free and immobilized enzymes were assessed on their respective enzymatic activities and for 17α-ethinylestradiol (EE2) degradation. The optimum pH conditions concerning laccase activity ranged from 3.6 to 4.6, while temperature ranged between 30 °C and 50 °C for both free and immobilized enzyme. Laccase produced using PD broth presented greater storage stability and thermal stability than that of PDM. Best EE2 removals were of 79.22% and 75.00% for the free and immobilized enzymes, respectively. Removal rates were assessed during 8 h at pH 5. The removal of 17α-ethinylestradiol was stabilized in the fourth cycle of use. Results imply that immobilization promoted stability towards pH and temperature variations, although media played a decisive role in the enzymatic activity. Both free and immobilized laccases of P. ostreatus were able to degrade EE2, whereas immobilized laccase in PDM medium presented possible reuse applicability, albeit removal was not optimal when compared to other reports.

Cupuaçu (Theobroma grandiflorum) residue and its potential application in the bioremediation of 17-Α-ethinylestradiol as a Pycnoporus sanguineus laccase inducer.

Bioremediation is a strategy to mitigate environmental impacts of hazardous pollutants from anthropogenic sources. Natural byproducts, including agroindustrial wastes (AW) can be used to induce enzyme biosynthesis, leading up to enhancement of pollutants degradation process. Therefore, this study aimed to evaluate the use of cupuaçu, Theobroma grandiflorum AW as Pycnoporus sanguineus Laccase (Lac) inducer in order to promote 17-α-ethinylestradiol (EE2) bioremediation. The macro and micro-nutrients levels of cupuaçu AWs were evaluated in order to establish further correlations with enzymatic biosynthesis induction. The fungus was cultivated for 7 days in temperature of 28 ± 2 °C and agitation of 150 rpm. For bioremediation, Lac enzymatic extract was added to EE2 solution (10 µg mL-1) and the percentage of removal was evaluated by HPLC after 1-24 hr of reaction. At optimized conditions, the enzyme extract production was remarkably enhanced by adding only 1% (w/v) of cupuaçu AW. Lac activity reached 1642 U mL-1 on the 6th day of culture, which was higher than positive control (511 U mL-1). 86% of EE2 removal was reached after 4 hr, and after 8 hr of reaction, 96.5% was removed. Analysis by direct infusion in MS-ESI-TOF exhibited intermediary compounds formed by radical hydroxilation.

Endocrine disruption in Sphoeroides testudineus tissues and sediments highlights contamination in a northeastern Brazilian estuary.

In recent decades, considerable attention has been devoted to endocrine disruptor chemicals (EDC) and studies on fish feminization have increased throughout the years as a key signal for aquatic environmental contamination. The input of domestic sewage into water reservoirs is common in South American countries, especially in cities that experienced rapid population growths and unplanned urbanization. This study aimed at characterizing morphofunctional parameters of the tropical fish Sphoeroides testudineus and investigating the potential occurrence and effects of endocrine disruptors in the Pacoti River (Ceará, Brazil), often considered a reference site. After collection from the field, fish were measure/weighted and desiccated for gender identification (males, females, and undifferentiated), gonadal histology, and vitellogenin expression. From the biometric analysis, undifferentiated fish showed lower weight and length than female and male fish, although no differences in the condition index were observed. The gonadal weight of undifferentiated fish was significantly lower than those of females and males. Although this pattern was observed, gonadosomatic index (GSI) showed a different pattern, with differences being observed just between males and the other two groups (females and undifferentiated). Vitellogenin (VTG) expression was detected in many mature male and undifferentiated fish, indicating endocrine disruption. In addition, several EDCs (estrone, 17α-estradiol, 17ß-estradiol, 17α-ethinylestradiol, diethylstilbestrol, and estriol) were identified and quantified in sediments from the sampling site. These results were unexpected and indicative that the Pacoti River is impaired by estrogenic contamination.

Removal of 17α-ethinylestradiol from a sterile WC medium by the cyanobacteria Microcystis novacekii.

An unprecedented investigation dealing with the removal of 17α-ethinylestradiol (EE2, a contraceptive hormone) by the cyanobacteria Microcystis novacekii (a species that is abundant and easily accessible in Brazilian lakes) from a sterile WC medium is described herein. The results indicated that whereas EE2 experienced insignificant spontaneous degradation, Microcystis novacekii was capable of removing ca. 65% of the hormone from the culture medium. Furthermore, no metabolites were detected at the concentration levels evaluated (0.10 to 0.17 mg L(-1)) as verified by the use of GC-MS, a quite sensitive analytical technique, and adequate pre-concentration procedures (SPME and liquid extraction). Elucidative experiments, including an appropriate cell lyses procedure, indicated that EE2 was likely accumulated within the cells (bioaccumulation) rather than adsorbed on the cellular membrane (biosorption). Moreover, the intra- and extracellular contents of EE2 were shown to be roughly complementary. Finally, the species was found to be highly tolerant to the hormone as its growth rates were higher in the test than in the control experiments. All these findings, therefore, point to the use of Microcystis novacekii as a potential agent to treat effluents contaminated with EE2.

Influence of operational parameters (sludge retention time and hydraulic residence time) on the removal of estrogens by membrane bioreactor.

INTRODUCTION: This paper deals with the removal of two natural estrogens, estrone (E1) and 17ß-estradiol (E2) and a synthetic one 17α ethinylestradiol (EE2) from wastewater in a laboratory-scale membrane bioreactor (MBR). MATERIALS AND METHODS: The effects of both solid retention time (SRT) and hydraulic residence time (HRT) were studied using synthetic wastewater in the MBR. At 35, 45, 60, 75, and 95 days, SRT was studied. The HRT was varied in the range of 7-12 h. RESULTS: The results showed that the increases in HRT and SRT enhanced the biodegradation process after adaptation to microorganisms. At HRT of 12 h, the estrogen removals were close to 100% in the MBR. The highest estrogen removals were obtained at SRT of 60 days. Continuous tests showed a linear relationship between nitrification and estrogen removal rates. CONCLUSIONS: The most biodegradable compound was the E2. The membrane fouling rates increased with the decreased of SRT and HRT. Optimal process conditions in this work was obtained at the SRT and HRT of 60 days and 12 h, respectively, with high efficient of estrogen removal, nitrification efficiencies, as well as a minimum membrane fouling rate.

Galactosamine prevents ethinylestradiol-induced cholestasis.

Ethinylestradiol (EE) induces intrahepatic cholestasis in experimental animals being its derivative, ethinylestradiol 17beta-glucuronide, a presumed mediator of this effect. To test whether glucuronidation is a relevant step in the pathogenesis of cholestasis induced by EE (5 mg/kg b.wt. s.c. for 5 consecutive days), the effect of simultaneous administration of galactosamine (200 mg/kg b.wt. i.p.) on biliary secretory function was studied. A single injection of this same dose of galactosamine was able to decrease hepatic UDP-glucuronic acid (UDP-GA) levels by 85% and excretion of EE-17beta-glucuronide after administration of a tracer dose of [3H]EE by 40%. Uridine (0.9 g/kg b.wt. i.p.) coadministration reverted the effect of galactosamine on hepatic UDP-GA levels and restored the excretion of [3H]EE-17beta-glucuronide. When administered for 5 days, galactosamine itself did not alter any of the serum markers of liver injury studied (aspartate aminotransferase, alanine aminotransferase, and alkaline phosphatase) or biliary secretory function. When coadministered with EE, galactosamine partially prevented the impairment induced by this estrogen in total bile flow, the bile-salt-independent fraction of bile flow, basal bile salt secretion, and the secretory rate maximum of tauroursodeoxycholate. Uridine coadministration partially prevented galactosamine from exerting its anticholestatic effects. In conclusion, galactosamine administration partially prevented EE-induced cholestasis by a mechanism involving decreased UDP-GA availability for subsequent formation of EE 17beta-glucuronide. The evidence thus supports the hypothesis that EE 17beta-glucuronide is involved in the pathogenesis of EE cholestasis.

Clinical and metabolic aspects of the continuous use of a contraceptive association of ethinyl estradiol (30 microg) and gestodene (75 microg).

This open, prospective, noncomparative study evaluated clinical and metabolic aspects of the use of a contraceptive combination of ethinyl estradiol (30 microg) and gestodene (75 microg) continuously for 24 weeks in 45 women aged 25 +/- 3.7 years. No alterations in weight or blood pressure were observed. Few side effects were recorded. Amenorrhea rates increased from the fourth month of observation onwards, reaching 81.2% by week 24. A reduction in the levels of cholesterol and LDL and an increase in HDL and triglycerides were observed. Insulin levels increased but not significantly, while levels of glycemia remained unchanged. Levels of antithrombin III, fibrinogen and plasminogen activator inhibitor-1 (PAI-1) increased, whereas a reduction was observed in proteins C and S and in prothrombin time (PT). Activated partial thromboplastin time (APTT) remained unchanged. The treatment was associated with satisfactory clinical effects, high rates of amenorrhea after the third treatment cycle, and resulted in metabolic changes similar to those encountered during the classic use of contraceptive pills with monthly interruption for withdrawal bleeding.

Ursodeoxycholate reduces ethinylestradiol glucuronidation in the rat: role in prevention of estrogen-induced cholestasis.

Ethinylestradiol (EE) administration (5 mg/kg, s.c., daily for 5 days) to rats leads to cholestasis, and its derivative EE 17beta-glucuronide is a likely mediator of this effect. Coadministration of ursodeoxycholate (UDC) was shown to prevent ethinylestradiol-induced cholestasis. The aim of this study was to evaluate the inhibitory effect of UDC on EE glucuronidation in vivo and in vitro as a potential mechanism to explain UDC protection. UDC treatment (25 mg/kg, i.p., daily for 5 days) decreased the biliary excretion of EE 17beta-glucuronide in bile after administration of a trace dose of [3H]EE and reduced microsomal EE 17beta-glucuronidation activity by 20% and expression of UGT2B1, one of the enzymes involved in EE conjugation, by 30%. Glucuronidation kinetic studies were performed in vitro using normal microsomes and isolated hepatocytes in the presence of tauroursodeoxycholate (TUDC), the major endogenous derivative of UDC in the rat. Kinetic enzymatic studies in microsomes showed a noncompetitive inhibition of EE 17beta-glucuronidation by TUDC, which was unique for this bile salt since other endogenous bile salts such as taurocholate, taurochenodeoxycholate, or taurodeoxycholate did not affect the enzyme activity. Studies in isolated hepatocytes confirmed the inhibitory effect of TUDC on EE glucuronidation and indicated that TUDC can reach the enzyme active site in intact cells. In conclusion, both in vivo and in vitro experiments indicate that UDC decreased the metabolic pathways involved in EE glucuronidation, hence decreasing the formation of the cholestatic derivative EE 17beta-glucuronide.

En anticoncepción hormonal oral, ¿más o menos estrógenos? / Hormone oral contraceptives: more or less estrogens?

La disponibilidad de dos dosis de etinil estradiol (20 y 30 mcg) asociado a desogestrel (150 mcg), permite mayor elasticidad en la prescripción. En la experiencia del autor hay indicaciones preferentes para una y otra dosis: La de 20 mcg: a) en adolescentes sin acné; b) en personas delgadas, de menos de 60 Kg; c) en usuarias de busto grande y sangrados abundantes; d) en pacientes obesas que han alcanzado su peso ideal; e) en usuarios de 35 o más años, apróximas a la menopausia y aún en el corto plazo postmenopausia, si son asintomáticas. La de 30 mcg: a) en adolescentes con acné; b) en pacientes con busto pequeño y sangrados cíclicos escasos; c) en usuarias de más de 60 Kg.; d) en las obesas en tratamiento, mientras alcanzan el peso ideal

La combinación desogestrel etinil estradiol y el peso corporal excedido: resultados actuales / Desogestrel-ethynil estradiol combination and overweight: current data

Se revisan los efectos de diversos progestágenos combinados con etinil estradiol sobre el peso corporal. Un nuevo grupo de pacientes obesas sometidas a tratamiento reductor, que además consumían anticonceptivos hormonales combinados (150 mg desogestrel-etinil estradiol, 30 mcg.) fue estudiado en condiciones similares a las del primer grupo (1992), obteniendose resultados similares, muy satisfactorios; bajan de peso hasta alcanzar el peso ideal, comprobándose que el anticonceptivo que utilizan, desogestrel-etinil estradiol, no interfiere con el tratamiento reductor

Efectos clínicos y del metabolismo lipídico de un anticonceptivo monofásico a base de gestodeno y etinilestradiol / Clinical side effects and in lipid metabolism of a monofasic contraceptive containing gestodene and ethinylestradiol

Se realizó un estudio clínico para evaluar la efectividad y efectos colaterales clínicos y en metabolismo lipídico de un preparado anticonceptivo hormonal combinado que contiene 75 mcg. de gestodeno y 30 mcg. de etinilestradiol. Se incluyeron 30 mujeres mexicanas en edad reproductiva, las que se estudiaron en periodo de 12 meses acumulando un total de 360 meses-mujer. El preparado fue eficaz puesto que no se presentaron embarazos en el lapso estudiado. Los efectos clínicos secundarios fueron más leves y menos frecuentes a los observados con otros anticonceptivos orales disponibles. En lo referente al metabolismo de los lípidos no se observaron los cambios adversos más frecuente detectados con el empleo de progestágenos previamente disponibles. Al igual que otros estudios, este trabajo concluye que el nuevo preparado, gestágeno más etinilestradiol, es altamente eficaz como anticonceptivo, con baja incidencia de efectos colaterales y debido a su inteferencia sobre metabolismo lipídico, es particularmente indicado para casos de alto riesgo de desarrollar patología asociada a alteraciones en el metabolismo de lis lípidos

Efectos de la combinación gestodeno etinilestradiol sobre el endometrio / Effects of the gestodene ethynilestradiol combination in endometrium

Con la hipótesis de trabajo de que la efectividad anticonceptiva comprobada de la mezcla de 75 mcg. de gestodeno con 30 mcg. de etinilestradiol es debida en parte de los cambios morfológicos e histoquímicos que inducen en el endometrio, se administró esta medicación durante 21 días de cada ciclo a partir del 1er. día durante seis ciclos a 32 pacientes sanas, ovulatorias y con fertilidad previa comprobada que solicitaron anticoncepción hormonal y aceptaron participar en el estudio. Como parte de la vigilancia de su evolución, se les tomaron biopsias de endometrio en fase progestacional en el ciclo previo y, en el sexto ciclo de tratamiento, al día siguiente de terminar la toma de esteroides, a diez de ellas escogidas al azar. Los endometrios se estudiaron mediante tención de hamatoxilina-eosina y por los métodos histoquímicos para determinar glucógeno y ácidos nucléicos y las imágenes se juzgaron con los criterios establecidos en trabajos previos para este tipo de estudios. Los cambios morfológicos observados fueron semejantes a los ya conocidos para otras mezclas anticonceptivas, pero en la histoquímica se observó alteración del glucógeno semejante a la que producen los esteroides 19-nor, mientras que los ácidos nucleicos semejan más a lo encontrado con esteroides 17-acetoxi. Se confirma que la mezcla en estudio sí modifica la biología endometrial.