Tissue-specific uptake and bioconcentration of the oral contraceptive norethindrone in two freshwater fishes.

The environmental presence of the oral contraceptive norethindrone (NET) has been reported and shown to have reproductive effects in fish at environmentally realistic exposure levels. The current study examined bioconcentration potential of NET in fathead minnow (Pimephales promelas) and channel catfish (Ictalurus punctatus). Fathead minnows were exposed to 50 µg/l NET for 28 days and allowed to depurate in clean water for 14 days. In a minimized 14-day test design, catfish were exposed to 100 µg/l NET for 7 days followed by 7-day depuration. In the fathead test, tissues (muscle, liver, and kidneys) were sampled during the uptake (days 1, 3, 7, 14, and 28) and depuration (days 35 and 42) phases. In the catfish test, muscle, liver, gill, brain, and plasma were collected during the uptake (days 1, 3, and 7) and depuration (day 14) stages. NET tissue levels were determined by gas chromatography-mass spectrometry (GC-MS). Accumulation of NET in tissues was greatest in liver followed by plasma, gill, brain, and muscle. Tissue-specific bioconcentration factors (BCFs) ranged from 2.6 to 40.8. Although NET has been reported to elicit reproductive effects in fish, the present study indicated a low potential to bioconcentrate in aquatic biota.

Are oral contraceptives a significant contributor to the estrogenicity of drinking water?

Recent observed feminization of aquatic animals has raised concerns about estrogenic compounds in water supplies and the potential for these chemicals to reach drinking water. Public perception frequently attributes this feminization to oral contraceptives (OCs) in wastewater and raises concerns that exposure to OCs in drinking water may contribute to the recent rise in human reproductive problems. This paper reviews the literature regarding various sources of estrogens, in surface, source and drinking water, with an emphasis on the active molecule that comes from OCs. It includes discussion of the various agricultural, industrial, and municipal sources and outlines the contributions of estrogenic chemicals to the estrogenicity of waterways and estimates that the risk of exposure to synthetic estrogens in drinking water on human health is negligible. This paper also provides recommendations for strategies to better understand all the potential sources of estrogenic compounds in the environment and possibilities to reduce the levels of estrogenic chemicals in the water supply.

Evaluation of analytical methodology for the detection of hormones and their attenuation during aquifer recharge and recovery cycles.

The hormones listed in the screening survey list 2 of the Unregulated Contaminant Monitoring Rule 3 (estrone, 17-ß-estradiol, 17-α-ethynylestradiol, 16-α-hydroxyestradiol (estriol), equilin, testosterone and 4-androstene-3,17-dione) were analyzed by liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). Two analytical methods were compared: EPA method 539 and the isotope dilution method. EPA method 539 was successfully utilized in river and drinking water matrices with fortified recoveries of 98.9 to 108.5%. Samples from the Hillsborough River reflected levels below the method detection limit (MDL) for the majority of the analytes, except estrone (E1), which was detected at very low concentrations (<0.5 to 1 ng L(-1)) in the majority of samples. No hormones were detected in drinking water samples. The isotope dilution method was used to analyze reclaimed and aquifer storage and recovery (ASR) water samples as a result of strong matrix/solid phase extraction (SPE) losses observed in these more complex matrices. Most of the compounds were not detected or found at relatively low concentrations in the ASR samples. Attenuation of 50 to 99.1% was observed as a result of the ASR recharge/recovery cycles for most of the hormones, except for estriol (E3). Relatively stable concentrations of E3 were found, with only 10% attenuation at one of the sites and no measureable attenuation at another location. These results have substantiated that while EPA method 539 works well for most environmental samples, the isotope dilution method is more robust when dealing with complex matrices such as reclaimed and ASR samples.

Rapid, sensitive colorimetric method for determination of ethinyl estradiol.

A colorimetric procedure, based on the formation of an azo dye by condensation of diazotized 5-chloro-2,4-dinitroaniline with ethinyl estradiol, was developed. An alkaline solution of ethinyl estradiol is reacted with the reagent, and the resulting color is measured at 450 nm. Absorbance versus concentration is linear up to 10 mug/ml; the lower limit of detection is 1 mug/ml under the conditions studied. Replicate analysis showed good agreement, and an average recovery of 99.6 +/- 0.3% was obtained for analyses of synthetic mixtures. Vitamins and minerals likely to be present along with ethinyl estradiol in certain geriatric formulations, as well as ordinary tablet excipients and coating materials, do not interfere with the precision of the method or development of the color. The method is applicable to progestin-estrogen preparations. Assay results on various single-component as well as contraceptive commercial samples are reported.

Quantitative analysis of ethynodiol diacetate and ethinyl estradiol/mestranol in oral contraceptive tablets by high-performance liquid chromatography.

A procedure is described for the assay of ethynodiol diacetate and ethinyl estradiol/mestranol by HPLC using two UV detectors at 210 and 280 nm. The system was acetonitrile 38% (v/v) in water as mobile phase on a 250 x 3.2-mm i.d. RP-2 column, with butylated hydroxytoluene as the internal standard. There was greater than 99% recovery from synthetic preparations and the coefficient of variation was greater than 2.0% for formulations.

Characterization of the novel progestin gestodene by receptor binding studies and transactivation assays.

Gestodene is a novel progestin used in oral contraceptives with an increased separation of progestogenic versus androgenic activity and a distinct antimineralocorticoid activity. This specific pharmacological profile of gestodene is defined by its pattern of binding affinities to a variety of steroid hormone receptors. In the present study the affinity of gestodene to the progesterone receptor (PR), the androgen receptor (AR), the glucocorticoid receptor (GR), the mineralocorticoid receptor (MR) and the estrogen receptor (ER) was re-evaluated by steroid binding assays and compared to those obtained for 3-keto-desogestrel and progesterone. The two synthetic progestins displayed identical high affinity to rabbit PR and similar marked binding to rat AR and GR, while progesterone showed high affinity to PR but only low binding to AR and GR. Furthermore, 3-keto-desogestrel exhibited almost no binding to MR, whereas gestodene, similar to progesterone, showed marked affinity to this receptor. In addition to receptor binding studies, transactivation assays were carried out to investigate the effects of gestodene on AR-, GR- and MR-mediated induction of transcription. In contrast to progesterone, which showed antiandrogenic activity, gestodene and 3-keto-desogestrel both exhibited androgenic activity. Furthermore, all three progestins exhibited weak GR-mediated antagonistic activity. In contrast to progesterone, which showed almost no glucocorticoid activity, gestodene and 3-keto-desogestrel showed weak glucocorticoid action. In addition, gestodene inhibited the aldosterone-induced reporter gene transcription, similar to progesterone, whereas unlike progesterone, gestodene did not induce reporter gene transcription. 3-Keto-desogestrel showed neither antimineralocorticoid nor mineralocorticoid action.

The effect of lactation on ovulation and fertility.

It has long been recognized that women who breastfeed their children have a longer period of amenorrhea and infertility following delivery than do those women who do not breastfeed. The length of postpartum amenorrhea is quite variable, and depends on several factors, including maternal age and parity, and the duration and frequency of breastfeeding. In general, it would appear that the more frequent and the longer the episodes of breastfeeding, the longer will be the period of anovulation, and the longer the period of infertility.

PIP: The effect of lactation on ovulation and fertility is discussed in relation to 7 factors: the duration of postpartum amenorrhea, the return of ovulation in the postpartum woman, the effect of breastfeeding on fertility, the physiologic basis for infecunity during lactation, contraceptive use during lactation (barrier methods, IUDs, and steroidal contraceptives), breastfeeding while pregnant, and tandem nursing. Women who breastfeed their children have a longer period of amenorrea and infertility following delivery than women who do not breastfeed. The length of postpartum amenorrhea varies greatly and depends on several factors, including maternal age and parity and the duration and frequency of breastfeeding. Due to the fact that there exists such individual variability in the duration of daily suckling, as well as the duration of the breastfeeding period, it is not possible to define within narrow limits the expected period of postpartum amenorrhea in lactating women. The return of menstruation is not necessarily the result of preceding ovulation in the postpartum woman. There is a wide range in the reports as to the occurrence of ovulation before 1st menstruation, ranging from 12-78%. In general, ovulation precedes 1st menstruation more frequently in those who do not nurse when compared to those who nurse. Breastfeeding has a demonstrable influence in inhibiting ovulation; it is not surprising that it has an inhibiting effect on fertility. According to Perez, during the first 3 months when a woman is nursing, there is higher security provided agaist conception than most contraceptives. After that time, the effect on fertility becomes uncertain and is determined by the frequency and duration of suckling and the time interval from delivery, and possibly maternal age, parity, nutrition. The physiologic basis for lactation infertility is not completely understood. During pregnancy, the level of circulating prolactin is greatly elevated. The elevated blood levels of prolactin begin at 8 weeks and rise to levels of 200 ng per ml at term. In lactating women, prolactin levels stay elevated, with spikes of increased secretion during and following suckling. The evideence points strongly to the fact that persistent hyperprolactinemia caused by breastfeeding postpartum results in an anovulatory or oligo-ovulatory state, and this results in relative infertility. It is appropriate to suggest other contraceptive methods to women who want to delay subsequent pregnancy because lactation alone is unreliable in preventing conception after the 9th week postpartum. There appears to be no contradindications to the use of the vaginal diaphragm or condom while breastfeeding. A report of added risk of uterine perforation in lactating women requires confirmation. The use of steroidal contraceptives while breastfeeding remains controversial.

Serum folate and Vitamin B12 levels in women using modern oral contraceptives (OC) containing 20 microg ethinyl estradiol.

OBJECTIVE: The effects of modern oral contraceptives (OC) on serum concentrations of folate and cobalamin are controversial. STUDY DESIGN: Case-control study on the cobalamin and folate status of 71 healthy female nulligravidae using "low dose" OC for >or=3 months and 170 controls. Factors interfering with vitamin metabolism were thoroughly controlled. Serum concentrations were measured by commercial assays. The results were evaluated using Mann-Whitney's U-test and chi(2) analysis. RESULTS: OC-users showed significantly lower concentrations of cobalamin than controls. The rates of women with reduced, normal, and elevated levels differed significantly. Nine users but no control had frank cobalamin deficiency without clinical symptoms. Folate levels did not differ between the groups. Vegetarian diet, smoking or obesity did not have a significant influence. CONCLUSIONS: Routine measurement of cobalamin or folate in women using "low dose" OC is not warranted. Vitamin supplementation or different contraceptive methods should be considered in women with pre-existing cobalamin deficiency or restrictive dietary habits.

Use and misuse of the term potency with respect to oral contraceptives.

PIP: As a result of studies suggesting an association between oral contraceptive (OC) use and breast cancer and cardiovascular events, there has been considerable discussion of the estrogenic and progestogenic potency of OC formulations. This article seeks to portray the state of the art and the validity of current potency estimates. The relative potency of 2 drugs is the ratio of doses have an equivalent effect when the dose/response curves are parallel. In such assessments, all OC constituents must be specified, standardized, and replicated. Tests measuring control of cyclic uterine bleeding and efficiency of ovulation inhibition are of primary importance for the clinical assessment of an OC. The synergism and antagonism of estrogens and progestogens have been demonstrated in animal studies. Progestational compounds may have estrogenic, antiestrogenic, androgenic, antiandrogenic, and corticoid activities as well as progestational activity. Moreover, the assay of such activity may have no clinical correlate. Even when a clinical endpoint such as the delay of menses is used, the results are difficult to interpret. In short, none of the indices proposed to assess progestational potency in humans has led to the development of even remotely satisfactory bioassay systems, for both technical and practical reasons. It appears that reliable data will become available only through sound epidemiologic studies of substantial magnitude.^ieng

Determination of ethinylestradiol and norethisterone in an oral contraceptive capsule by reversed-phase high performance liquid chromatography.

A specific and sensitive analytical method is described for the simultaneous determination of ethinylestradiol and norethisterone in a capsule formulation. These steroids, commonly used in oral contraceptives, were extracted from the capsules with acetonitrile and tetrahydrofuran. The steroids were then quantitated with a high performance liquid chromatograph using a ODS reversed-phase column and a ternary solvent system of water, acetonitrile, and tetrahydrofuran as the mobile phase. Several solvent systems for the mobile phase were examined using various C18 columns. The k' values for several steroids are reported, together with column performance parameters. It was found that columns from different manufacturers had significantly different behaviors with respect to separation parameters for these steroids.

PIP: This is a description of the use of a reversed-phase HPLC (high performance liquid chromatography) system for the simultaneous analysis of both steroids, NET (norethisterone) and EE (ethinylestradiol), in a capsule form of combined OCs (oral contraceptives). The method is seen to provide a specific, quick, and accurate assay of the 2 most commonly used steroids. These 2 steriods were extracted from the capsules and quantitated with a HPLC using a bonded octadecylsilane reversed-phase column and a ternary solvent system of water, acetonitrile, and tetrahydrofuran as the mobile phase. Columns from different manufacturers behaved differently with respect to separation parameters for the 2 steroids. 2 problems inherent in the analytic method are: 1) the low ultraviolet molar absorptivity of EE; and 2) the low proportion of EE compared to NET. The described procedure can also be used for analysis of other forms of the combined steroids, e.g., tablets.

Gas-liquid chromatographic analysis of lynestrenol in contraceptive tablets.

A simple procedure for the estimation of lynestrenol in contraceptive tablets is described. The method consists of extraction of the hormone into dichloromethane followed by g.l.c. of the resulting solution. 3 alpha-Etiocholanol-17-one is demonstrated as a suitable internal reference standard.

PIP: Current methods used to estimate the lynestrenol content of oral contraceptive tablets present drawbacks usually due to the presence of mestranol, a synthetic estrogen, in the tablet. Gas-liquid chromatography (GLC) was found to be both a specific and simple procedure for lynestrenol determination. Preliminary studies using solutions of 3-alpha-etiocholanol-17-one and lynestrenol showed complete separation of peaks and a relatively constant peak area ratio making the former substance an ideal internal reference standard (Figure 1; Table 1). In addition, solutions containing various amounts of lynestrenol, mestranol and etiocholanol separated completely using GLC. Prepared mixtures of lynestrenol-mestranol and some commercial tablets were combined with 5 ml of the internal standard solution (5 mg 3-alpha-etiocholanol-17-one/1 ml dichloromethane), centrifuged, chromatographed and compared to a previously plotted standard curve. In all cases, the recovery of lynestrenol is reproducible and almost complete (Tables 2-3) making GLC a desirable method for lynestrenol analysis.

[Classification of oral contraceptives and its practical application to choice of prescriptions]. / Essai de classification des contraceptifs oraux et application pratique au choix des prescriptions.

The authors have tried to classify the oral contraceptives at present in use throughout the world by their respective levels of oestrogenic and progestogenic components, taking into consideration also the relative strength of each product in use. They have based this classification on their personal experience of the several oral contraceptive products and on a profound study of the international literature. In this way it is possible to define a place for each oral contraceptive so that it can be chosen for the best possible use of a particular product for the hormonal background of the person using the contraceptive. It is true that changes have to be made to work out the oral contraceptives used for masses of people, but on the other hand in countries where there is sufficient medical personnel and education so that it is possible to think out and to prescribe for an individual, it seems to us useful that the doctor who is prescribing should try to adjust the oral contraceptive to the hormonal climate of the woman seeking it. This is true not only for the first time a prescription is made but in cases where by the accident of failure to tolerate the product something must be done to correct the troubles that are so often found in the first months after starting to use contraceptive products. This study includes a general picture of all oral contraceptives in which the products have been classified according to the total dose of oestrogens administered and according to the proportions of progesterone to oestrogens which make up the hormonal climate of the product. On the other hand this study also includes a list of the observations to make in order to adapt the oral contraceptive to the side-effects shown after the first prescription. The authors have come to the conclusion that by adapting the choice of a particular oral contraceptive to the woman who is to receive it interruptions in taking the contraceptive because of side-effects should be avoided. So unwanted pregnancies will be avoided and also the termination of these unwanted pregnancies.

[Adverse effects of oral contraceptives]. / Bivirkninger ved brug af orale kontraceptiva

PIP: A study of 990 reports of side effects experienced by users of oral contraceptives between 1968-1974 is presented. The 3 most frequently reported side effects were thromboembolic distrubances, psychical disturbances, and weight gain. Thromboembolic distrubances were significantly more frequent with higher estrogen content. A correlation between estrogen content and psychical distrubances or weight gain could not be definitively shown due to changes in the use of the various brands of contraceptives and the decrease of reports about these side effects over the years. The frequency of side effects reported for selected single preparations were compared. A correlation was shown between the occurrence of weight gain and mental disturbance; the relationship showed no correlation to estrogen content and only a slight correlation to the gestagen component.^ieng

[Estrogens. Contraceptive therapy]. / Estrógenos. Terapia anticonceptiva.

To evaluate the competitive molecular phenomenon of Ethynyl-Estradiol (EE-2) from contraceptive formulations against the endogenous Estradiol (E-2) at the intra and the extracellular compartments, plasma and endometrial samples were simultaneously obtained on different days of the pseudomenstrual cycle from oral contraceptive users under EE-2+ Norgestrel (30 micrograms/+ 500 micrograms) and EE-2+ Norethindrone (50 micrograms + 1.0 mg) in order to quantify EE-2 & E-2. When measuring both molecules it was shown that the chronic administration of steroids regardless of the pharmacological action of the progestin component the lower content of EE-2 (30 micrograms) does not compete substantially at the circulating level permitting the cyclic fashion of the natural estradiol while at the endometrial compartment such phenomenon is not seen thus, a local infertility effect should be reconsidered to anticipate a different approach in the future of contraception.

PIP: 27 women participated in a study of systemic and endometrial estradiol concentrations in oral contraceptive (OC) users. 13 women aged 15-22 used OCs containing ethinyl estradiol 30 mcg and norgestrel 500 mg for 4-24 months, and 14 women aged 18-28 used OCs containing 50 mcg ethinyl estradiol and 1.0 mg norethindrone for 1-28 months. A peripheral blood sample and an endometrial biopsy were obtained at random from each woman on different cycle days to measure the concentration of endogenous estradiol. The ovarian response defined by endogenous plasma and endometrial estrogen levels differed greatly in the two groups. In the 30 mcg group, plasma estrogen levels were similar to those described in an ovulatory menstrual cycle, while this profile was not observed in the 50 mcg group. The endometrial estrogen profile described for women not using hormonal contraception was not observed in either of the two dose groups.

[Simultaneous determination using gas chromatography of mestranol and norethisterone in estrogen-progestins combination for oral use]. / Determinazione simultanea mediante gas-cromatografia del mestranolo e del noretisterone nelle associazioni estrogeno-progestiniche per uso orale.

PIP: The article describes a new method to determine the quantity of mestranol and of norethisterone in a combined oral contraceptive. Gas chromatography was used simultaneously to detect both agents, after they were extracted with ethyl acetate from the total compound of the tablet.^ieng

Hormonal contraception: current perspectives. Part I. An analysis of available agents.

PIP: Currently available forms of hormonal contraceptives with regard to their mechanism of action, effectiveness, and side effects are reviewed. The effectiveness of an oral contraceptive in preventing pregnancy is directly related to its ability to inhibit ovulation. Other effects are less important. The development of synthetic progestational agents made inhibition of ovulation possible without unacceptable side effects. The chemical structures of several estrogens are shown and their relationship to natural estrogen indicated. The chemical structures of synthetic progestational agents and their relationship to progesterone and testosterone are illustrated. Diethylstilbestrol is a nonsteroidal estrogenic compound with a potency of 1/25 of that of ethinyl estradiol. It is used only as a postcoital contraceptive for occasional use. Medroxyprogesterone acetate is a long-lasting injectable contraceptive agent. Available oral contraceptive preparations are listed by brand names, hormone content, and tablet amount. With combination compounds a 28-day routine of therapy has been established. In recently developed compounds the amount of hormone used has been reduced so that side effects are less. The pregnancy rate is less than .1%. The very low-dose preparations have a higher pregnancy rate and an increased incidence of intermenstrual bleeding. The sequential-type oral contraceptive is no longer available. The progestin-only type contains no estrogen, and they are taken continuously. They do not prevent ovulation. Their contraceptive effect is by alterations in cervical mucus and endometrial or tubal physiology. The pregnancy rate is relatively high. No single preparation is considered as suitable for all women. Patients with varicose veins or previous thromboembolic phenomena should not use any oral contraceptive. The combination preparations are most suitable for the majority of women.^ieng

Contraceptive options and their associated estrogenic environmental loads: relationships and trade-offs.

This work explores the relationships between a user's choice of a given contraceptive option and the load of steroidal estrogens that can be associated with that choice. Family planning data for the USA served as a basis for the analysis. The results showed that collectively the use of contraception in the USA conservatively averts the release of approximately 4.8 tonnes of estradiol equivalents to the environment. 35% of the estrogenic load released over the course of all experienced pregnancies events and 34% the estrogenic load represented by all resultant legacies are a result of contraception failure and the non-use of contraception. A scenario analysis conducted to explore the impacts of discontinuing the use of ethinylestradiol-based oral contraceptives revealed that this would not only result in a 1.7-fold increase in the estrogenic loading of the users, but the users would also be expected to experience undesired family planning outcomes at a rate that is 3.3 times higher. Additional scenario analyses in which ethinylestradiol-based oral contraceptive users were modeled as having switched entirely to the use of male condoms, diaphragms or copper IUDs suggested that whether a higher or lower estrogenic load can be associated with the switching population depends on the typical failure rates of the options adopted following discontinuation. And, finally, it was estimated that, in the USA, at most 13% of the annual estrogenic load can be averted by fully meeting the contraceptive needs of the population. Therefore, while the issue of estrogen impacts on the environment cannot be addressed solely by meeting the population's contraceptive needs, a significant fraction of the estrogenic mass released to environment can be averted by improving the level with which their contraceptive needs are met.

Degradation of the potential rodent contraceptive quinestrol and elimination of its estrogenic activity in soil and water.

Quinestrol has shown potential for use in the fertility control of the plateau pika population of the Qinghai-Tibet Plateau. However, the environmental safety and fate of this compound are still obscure. Our study investigated degradation of quinestrol in a local soil and aquatic system for the first time. The results indicate that the degradation of quinestrol follows first-order kinetics in both soil and water, with a dissipation half-life of approximately 16.0 days in local soil. Microbial activity heavily influenced the degradation of quinestrol, with 41.2% removal in non-sterile soil comparing to 4.8% removal in sterile soil after incubation of 10 days. The half-lives in neutral water (pH 7.4) were 0.75 h when exposed to UV light (λ = 365 nm) whereas they became 2.63 h when exposed to visible light (λ > 400 nm). Acidic conditions facilitated quinestrol degradation in water with shorter half-lives of 1.04 and 1.47 h in pH 4.0 and pH 5.0 solutions, respectively. Moreover, both the soil and water treatment systems efficiently eliminated the estrogenic activity of quinestrol. Results presented herein clarify the complete degradation of quinestrol in a relatively short time. The ecological and environmental safety of this compound needs further investigation.

Initial photocatalytic degradation intermediates/pathways of 17alpha-ethynylestradiol: effect of pH and methanol.

This study investigated the photocatalytic degradation of the synthetic oral contraceptive 17alpha-ethynylestradiol (EE2). Particular attention was paid on the effects of pH and the co-solvent methanol on the degradation intermediates of EE2. Twelve intermediates were identified, and several intermediates reported herein have not been found in previous studies. The degradation efficiency of EE2 and the number of identified intermediates decreased evidently at pH 3 and in the presence of methanol at pH 7. Three photocatalytic degradation pathways were proposed: The transformation of the phenolic moiety (pathway I) is the primary initial reaction pathway; the initial photocatalytic degradation in the aliphatic carbon linked to the aromatic ring (pathway II) only took place at pH 7; the isomerization of EE2 (pathway III) could occur only in the presence of methanol at pH 7. Results from this study underscore the importance of photocatalytic degradation on the removal of estrogenic activity mainly expressed by the phenol moiety of EE2.