Ethinylestradiol and Levonorgestrel as Active Agents in Normal Skin, and Pathological Conditions Induced by UVB Exposure: In Vitro and In Ovo Assessments.

The link between melanoma development and the use of oral combined contraceptives is not fully elucidated, and the data concerning this issue are scarce and controversial. In the present study, we show that the components of oral contraceptives, ethinylestradiol (EE), levonorgestrel (LNG), and their combination (EE + LNG) ± UVB (ultraviolet B radiation) induced differential effects on healthy (human keratinocytes, fibroblasts, and primary epidermal melanocytes, and murine epidermis cells) and melanoma cells (human-A375 and murine-B164A5), as follows: (i) at low doses (1 µM), the hormones were devoid of significant toxicity on healthy cells, but in melanoma cells, they triggered cell death via apoptosis; (ii) higher doses (10 µM) were associated with cytotoxicity in all cells, the most affected being the melanoma cells; (iii) UVB irradiation proved to be toxic for all types of cells; (iv) UVB irradiation + hormonal stimulation led to a synergistic cytotoxicity in the case of human melanoma cells-A375 and improved viability rates of healthy and B164A5 cells. A weak irritant potential exerted by EE and EE + LNG (10 µM) was assessed by the means of a chick chorioallantoic membrane assay. Further studies are required to elucidate the hormones' cell type-dependent antimelanoma effect and the role played by melanin in this context.

Reproductive drugs and environmental contamination: quantum, impact assessment and control strategies.

Industrial and municipal solid wastes, noise, pesticides, fertilizers and vehicular emission are visible pollutants responsible for environmental contamination and ill-effects on health of all living systems. But, environmental contamination due to drugs or medicines used for different purposes in humans and animals goes unseen largely and can affect the health of living system severely. During the last few decades, the usage of drugs has increased drastically, resulting in increased drug load in soil and water. Contraceptive and fertility drugs are extensively and effectively used in humans as well as animals for different purposes. Usage of these reproductive drugs in humans is increased manifold to manage reproductive problems and/or for birth control with changing lifestyles. These drugs are excreted in urine and faeces as metabolite or conjugated forms, leading to contamination of water, milk and animal produce, which are consumed directly by humans as well as animals. These drugs are not eliminated even by water treatment plant. Consumption of such contaminated water, milk, meat and poultry products results in reproductive disorders such as fertility loss in men and increase risk of different types of cancers in humans. Therefore, assessment of impact of environmental contamination by these drugs on living system is of paramount importance. The purpose of this review article is to provide a comprehensive analysis of various research and review reports on different contraceptive and fertility drugs used in human and animals, their occurrence in the environment and their ill-effects on living systems. The approaches to control this invisible menace have also been proposed.

Diacerein inhibits Estradiol-benzoate induced cervical hyperkeratosis in female rats.

Cervical hyperkeratosis is a common gynecological lesion and usually caused by inflammation or trauma. We investigated the effect of Diacerein on Estradiol benzoate-induced cervical hyperkeratosis. Diacerein (50mg/kg/day) was given orally to rats for 4 weeks in the presence or absence of cervical hyperkeratosis induced by intramuscular injection of Estradiol benzoate (60µg/100g) 3 times per week for 4 weeks. We measured the serum levels of total cholesterol, uterine weights, uterine tissue malondialdehyde, total nitrites, superoxide dismutase activity, caspase-3, interleukin-1b immunoexpression and histopathology. Our results showed that Estradiol benzoate succeeded to induce cervical hyperkeratosis which was detected by typical histopathological changes. In addition; there was significant reduction in superoxide dismutase levels and caspase-3 immunoexpression but significant increase in serum total cholesterol, malondialdehyde, total nitrites and interleukin-1b immunoexpression. Diacerein could improve all measured parameters to normal levels. It markedly prevented cervical hyperkeratosis through its anti-inflammatory (IL-1b receptor inhibitor), antioxidant and anti-apoptotic effects.

Drospirenone intake alters plasmatic steroid levels and cyp17a1 expression in gonads of juvenile sea bass.

Drospirenone (DRO) is one of the most widely used progestins in contraceptive treatments and hormone replacement therapies. The pharmacokinetics and potential toxicological effects of DRO were investigated in juvenile sea bass (Dicentrarchus labrax) exposed through the diet (0.01-10 µg DRO/g) for up to 31 days. DRO was detected in the blood (4-27 ng/mL) of fish exposed to the highest concentration, with no significant bioaccumulation over time and no alteration of hepatic metabolizing enzymes, namely, CYP1A and CYP3A-catalysed activities and UDP-glucuronyltransferase (UGT). Pregnenolone (P5), progesterone (P4), 17α-hydroxyprogesterone (17P4), 17α-hydroxypregnenolone (17P5), androstenedione (AD) and testosterone (T) were determined in plasma and gene expression of cyp17a1, cyp19a1a and cyp11ß analysed by qRT-PCR in gonads. The significant increase in plasmatic levels of 17P5, 17P4 and AD detected after 31 days exposure to 10 ng DRO/g together with the increased expression of cyp17a1 in females evidence the ability of DRO to alter steroid synthesis at low intake concentrations (7 ng DRO/day). However, the potential consequences of this steroid shift for female reproduction remain to be investigated.

Does metformin improve in vitro maturation and ultrastructure of oocytes retrieved from estradiol valerate polycystic ovary syndrome-induced rats.

BACKGROUND: Metformin decreases polycystic ovary syndrome (PCOS) symptoms, induces ovulation, and may improve developmental competence of in vitro oocyte maturation. This study was designed to define the effects of metformin on the characteristics of in vitro oocyte maturation in estradiol valerate (EV) PCOS-induced rats. METHODS: Forty-five adult female Sprague-Dawley rats were randomly divided into control; sham and PCOS-induced (treated by a single dose of estradiol valerate, 4 mg/rat, IM) groups. The body weight was measured weekly for 12 weeks. At the end of week 12, the serum levels of testosterone, estrogen, progesterone, LH, and FSH and blood glucose of all the rats were measured. About 380 cumulus oocyte complexes (control, 125; sham, 122; PCOS-induced rats, 133) were incubated in Ham's F10 in the absence and/or presence of metformin (M 5(-10)) for 12, 24, 36, and 48 h. The cumulus cells expansion and nuclear and cytoplasmic maturation of the oocytes was evaluated using 1 % aceto-orcein staining, and transmission electron microscopy (TEM). RESULTS: No significant differences were observed in the body weight of the rats. The serum level of testosterone was reduced, and progesterone and LH were significantly increased in the PCOS-induced rats (p < 0.05). However, no significant differences were observed in the serum levels of estrogen and FSH among the groups. Blood glucose level was higher in the PCOS-induced rats than control, (p < 0.01). The expansion of cumulus cells was observed in the metformin-treated oocytes. The oocytes retrieved from PCOS-induced rats show a stage of meiotic division (GVBD, MI, A-T, and MII) in 57.12 % of metformin-untreated and fairly significantly increased to 64.28 % in metformin-treated oocytes, (p < 0.05), but no differences were observed in the MII stage within groups. The redistribution of some cytoplasmic organelles throughout the ooplasm, particularly the peripheral cortical granules, was defined in the metformin-treated oocytes. CONCLUSIONS: Single dose of EV can creates a reversible PCO adult rat model. Metformin enhances the COCs to initiate meiotic resumption at the first 6 h of IVM. In our study the metformin inability to show all aspects of in vitro oocyte maturation and may be resulted from deficiency of EV to induce PCOS.

Synthesis, Cytotoxic and Contraceptive Activity of 6,8,9-Trihydroxy-2-methyl-2H-naphtho[2,3-b]pyran-5,10-dione, a Pigment of Echinothrix diadema, and its Analogs.

6,8,9-Trihydroxy-2-methyl-2H-naphtho[2,3-b]pyran-5,10-dion, a pigment of the sea urchin Echinothrix diadema, and six analogs were synthesized. The cytotoxic activity and contraceptive properties of the synthesized pyranonaphthazarins have been investigated using the sperm and eggs of the sea urchin Strongylocentrotus intermedius.

Role of macrophage secretions on rat polycystic ovary: its effect on apoptosis.

Polycystic ovarian syndrome is the most common endocrine disorder among women of reproductive age. Little is known about its etiology, although the evidence suggests an intrinsic ovarian abnormality in which endocrine, metabolic, neural and immune factors would be involved. In this work, the effects of macrophage (MO) secretion on ovarian apoptosis in a polycystic ovary syndrome rat model (PCO rat) induced by estradiol valerate are studied. Spleen MO secretions were used to stimulate ovaries and ovarian interstitial and granulosa cells from both PCO and control rats. Ovarian hormones and prostaglandin E2 (PGE2) were measured by RIA; ovarian mRNA levels of Bax, Bcl2 and NFkB by RT-PCR; and ovarian inducible nitric oxide synthase (iNOS) by western blot. The number of apoptotic cells was evaluated by TUNEL. In the PCO ovary, the MO secretions from PCO rats increased the Bax and NFkB mRNA expressions and increased TUNEL staining in both granulosa and theca cells. In addition, the PCO MO secretions produced a decrease of nitric oxide release, iNOS protein level and PGE2 content in the PCO ovary, and it also induced an increase of androstenedione production by PCO interstitial cells, in comparison with control MO secretions. Considering these results and knowing that testosterone stimulates tumour necrosis factor-α production by PCO MO modifying ovarian response by increasing androstenedione, it is reasonable to suggest that the increase of androgens stimulated in ovarian cells by PCO MO secretions could in turn stimulate the cytokine production from MO, thus maintaining an apoptotic vicious cycle in the PCO ovary.

Comparison of early morphological and molecular changes induced by 17-alpha-methyltestosterone and estradiol benzoate in the rat ovary.

Repeated exposure to 17-α-methyltestosterone (17MT) and estradiol benzoate (EB) for 28 or 90 days in rats induce similar ovarian atrophy. The objective of the present work was to identify and compare the early effects induced by 17MT and EB on the ovary using molecular and histopathological tools. Female rats were evaluated after 1, 3 or 7 days following an oral exposure by gavage at a daily dose of 600 mg/kg/day for 17MT and 5 mg/kg/day for EB. All animals were found to be acyclic after 3 or 7 days of treatment with 17MT and EB. Histopathological changes were present in the ovary, uterus, vagina and mammary gland after both treatments. Ovarian atrophy known as the long term effect of 17MT and EB was not yet detected after 7 days of treatment. But non regressive corpora lutea and cystic follicles were identically observed in the ovary of 17MT and EB treated females. Both compounds induced a decrease of LH transcripts together with an increase of plasma progesterone and prolactin levels. Differences in the profile of regulation of the aromatase were noted after 1 and 3 days of treatment in 17MT treated animals (upregulated) when compared to EB treated animals (downregulated). In summary, we have shown that despite the different nature of hormonal activity, EB and 17MT induce very early endocrine perturbation which presents several similarities. Our work indicated that the detection of early key hormonal markers in short term studies can help to predict the adverse long term effects on target tissues.

Antifertility and fetotoxic activities of Acanthus montanus aqueous extract in Wistar rats.

Acanthus montanus T. Anderson (Acanthaceae) possesses several medicinal properties; it is used in Cameroon as a folk medicine to treat pain, inflammation and threatened abortion. The aim of this study was to determine the effect of A. montanus aqueous extract on the estrous cycle pre- and postimplantation in rats and its mechanism of action. The estrous cycles of Wistar rats were monitored before, during and after oral administration of distilled water (control) or aqueous extract (62.5, 125, 250, 500, 1000 mg/kg/day). Furthermore, pregnant rats received the above doses of aqueous extract on days 1-6 (preimplantation) or 6-15 (postimplantation) of gestation and were sacrificed on day 8 or 20 of pregnancy, respectively. Moreover, aqueous extract (500 and 1000 mg/kg/day) was given to ovariectomized rats in the presence or absence of exogenously administered estrogen and/or progesterone and uterine weight and deciduoma count were evaluated. The extract, irrespective of dose, reversibly prolonged the metestrous and occasionally the diestrous stages of the estrous cycle. The extract did not alter the uterine wet weight or deciduoma count, suggesting a lack of estrogenic and progestational effects. At 1000 mg/kg/day, the extract caused appreciable preimplantation losses of 36.8 +/- 6.5% (P < 0.05), while none of the doses caused postimplantation losses. The extract also caused delayed fetal growth.

Nanosilver: a nanoproduct in medical application.

Nanotechnology is a most promising field for generating new applications in medicine. However, only few nanoproducts are currently in use for medical purposes. A most prominent nanoproduct is nanosilver. Nanosilver particles are generally smaller than 100nm and contain 20-15,000 silver atoms. At nanoscale, silver exhibits remarkably unusual physical, chemical and biological properties. Due to its strong antibacterial activity, nanosilver coatings are used on various textiles but as well as coatings on certain implants. Further, nanosilver is used for treatment of wounds and burns or as a contraceptive and marketed as a water disinfectant and room spray. Thus, use of nanosilver is becoming more and more widespread in medicine and related applications and due to increasing exposure toxicological and environmental issues need to be raised. In sharp contrast to the attention paid to new applications of nanosilver, few studies provide only scant insights into the interaction of nanosilver particle with the human body after entering via different portals. Biodistribution, organ accumulation, degradation, possible adverse effects and toxicity are only slowly recognized and this review is focusing on major questions associated with the increased medical use of nanosilver and related nanomaterials.

Evaluation of novel biodegradable cyclic carbonate polyester copolymers for cytocompatibility using MRC-5 cells.

The objective of this work was to synthesize and characterize a novel series of biodegradable cyclic carbonate polyester copolymers based on lactide and 5-methyl-5-benzyloxy-carbonyl-1,3-dioxan-2-one (MBC). Two compositions were selected for characterization. One copolymer was based on a racemic mixture of 1-lactide with 15.4 mole % MBC and the other was based on 1-lactide with 8.2 mole % MBC. These polymers contain carboxylic acid moieties along the backbone that may be used for tethering bioactive agents, forming ionic crosslinks or be reacted with vinyl containing monomers to allow free radical crosslinking. The initial materials evaluated have the carboxylic acid functionalities blocked with benzene. These polymers and the de-blocked versions may have potential applications for hard and soft tissue scaffolds, control drug delivery matrixes or a variety of other applications in medicine. The copolymer samples were pressed into 7.0-mm diameter disk using a KBr press. The disks were then sterilized using U.V radiation under a laminar flow hood. After sterilization, the copolymer disks were submerged in 2 ml of media and placed in a CO2 regulated incubator at 37 degrees C. A total of six groups per phase (n = 7 test tubes per group) were used in this study. Test tubes in groups I and III were plated with MRC-5 and subsequently treated with media alone (controls). Test tubes in groups II and IV were plated with MRC-5 and subsequently treated with media before being introduced to copolymer samples. Cell number, as well as, biochemical markers such as protein and malondialdehyde (MDA) were determined at the end of the 24, 48 and 72-hour time periods. Representative test tubes were subjected to an H&E staining procedure for microscopic morphological evaluation. The results of this evaluation suggest that the exposure of both copolymers produced a non-cytotoxic environment with the MRC-5 cell line. Although both copolymers are non-cytotoxic, the sample having the higher MBC content is the preferred composition based upon MDA levels and morphological evaluations.

Antifertility activity of Derris brevipes variety coriacea.

Traditional physicians in and around Kotagiri village near Ootacamund, use a mixture of powdered roots of Cassia occidentalis, Derris brevipes variety coriacea and Justicia simplex to control female fertility. A mixture of powdered roots of these three plants, powdered root of Derris brevipes variety coriacea and its ethanolic extract were screened for antifertility activity in proven fertile female rats at 200 and 600 mg/kg body weight, respectively and given orally on D(1-7) of pregnancy. Both doses of the root powder of Derris brevipes variety coriacea showed 50% anti-implantation activity and also a significant reduction in the number of litters born. The ethanolic extract exhibited 40% anti-implantation activity when given orally at 600 mg/kg body weight. The rats, which continued their pregnancy, did not deliver any litters after their full term. Hence, the combined antifertility (anti-implantation and abortifacient) activity of the ethanolic extract was 100%. The results suggest that the ethanolic extract possesses more abortifacient type effect than the anti-implantation activity. The ethanolic extract also exhibited weak estrogenic activity when given alone and tested in immature ovariectomised female albino rats. But, when given along with ethinyl estradiol, it exhibited slight antiestrogenic activity. Histological and biochemical estimations were carried out to confirm this.

Contraceptive gossypol blocks cell-to-cell communication in human and rat cells.

Gossypol (a polycyclic lipophilic agent naturally present in cottonseed, known as a potent non-steroid antifertility agent and a non-specific enzyme inhibitor) irreversibly impaired the intercellular communication between homologous pairs of various cultured cells, from man or rat, involved (Sertoli or trophoblastic cells) or not involved (ventricular myocytes) in steroidogenesis, in a dose-dependent manner. In serum-free assays, a rapid junctional uncoupling occurred in non-cytotoxic conditions. At 5 microM (approximately twice the peak plasma concentration measured in human patients during chronic administration), gap junctional communication was interrupted within 4 to 10 min, without concomitant rise in the intracellular Ca2+ concentration. The latter importantly increased when gossypol treatment was prolonged (cytotoxic effect). The short term uncoupling effect of gossypol was prevented by serum proteins, but long-lasting treatments (48 h) with moderate concentrations (3 microM) elicited junctional uncoupling and impeded the in vitro differentiation of human trophoblasts.

Synthesis and antifertility activity of some new fluorine containing 2-([2-(fluoroaryl)-1H-indol-3- yl]methylene)hydrazinecarbothioamides and 2-(fluoroaryl)-([5-(substituted benzylidene)-4-oxo-2-thiazolidinylidene]hydrazone)- 1H-indole-3-carboxaldehydes.

New fluorine containing 2-(fluoroaryl)-1H-indole-3-carboxaldehydes have been synthesized and subjected to reaction with thiosemicarbazide to give corresponding 2-([2- (fluoroaryl)-1H-indol-3-yl]methylene)hydrazinecarbothiamides which were cyclized in the presence of chloroacetic acid, sodium acetate and substituted benzaldehydes to 2-(fluoroaryl)-([5- (substituted benzylidene)-4-oxo-2-thiazolidinylidene]hydrazone)- 1H-indole-3-carboxaldehydes as potential antifertility agents. In preliminary screening, 2-(4'-fluorophenyl)- ([5-(methylene-3,4-dioxyphenyl)-4-oxo-2- thiazolidinylidene]hydrazone)-1H-indole-3-carbocaldehyde exhibited pronounced antifertility activity. All these new compounds have been characterized by analytical and spectral (IR, PMR, MS) studies.

Facts about an implantable contraceptive: memorandum from a WHO meeting.

This Memorandum reviews the results of research undertaken in animals and human subjects on the implantable contraceptive, Norplant, and where relevant, its components, levonorgestrel and Silastic. Results from clinical trials, including effectiveness and side-effects, are evaluated and service delivery aspects commented on. The Memorandum concludes with a statement regarding the use of Norplant as an option for long-term reversible contraception.

Uses of available record systems in epidemiologic studies of reproductive toxicology.

The uses of available record systems in epidemiologic studies of reproductive toxicology are described with reference to New York State. The available record systems (and relevant reproductive end points) described include: a newborn screening program for metabolic diseases and hemoglobinopathies (relevant to point mutations); chromosome registries and prenatal cytogenetics (for chromosome anomalies); live birth certificates (for birth defects, birthweight, sex ratio, etc); fetal death certificates (for spontaneous fetal deaths); and a statewide cancer registry (for childhood cancers and transplacental carcinogenesis). The uses and limitations of these record systems are discussed, along with examples of their use in descriptive and analytic epidemiologic studies. Descriptive studies outlined include investigations of temporal and geographic trends in birth defects, birth weight, and fetal deaths, with reference to environmental questions (eg, Love Canal, nuclear power plants). Analytic studies described concern parental occupation in relation to specific birth defects (neural tube defects and Down syndrome) and maternal use of contraceptive drugs.

PIP: The uses of available record systems in epidemiologic studies of reproductive toxicology are described with reference to New York State. The available record systems (and relevant reproductive end points) described include a newborn screening program for metabolic diseases and hemoglobinopathies (relevant to point mutations); chromosome registries and prenatal cytogenetics (for chromosome anomalies); live birth certificates (for birth defects, birthweight, sex ratio, etc); fetal death certificates (for spontaneous fetal deaths); and a statewide cancer registry (for childhood cancers and transplacental carcinogenesis). The uses and limitations of these record systems are discussed, along with examples of their use in descriptive and analytic epidemiologic studies. Descriptive studies outlined include investigations of temporal and geographic trends in birth defects, birthweight, and fetal deaths, with reference to environmental questions (Love Canal, nuclear power plants). Analytic studies described concern parental occupation in relation to specific birth defects (neural tube defects and Down syndrome) and maternal use of contraceptives.