Dynamics of metabolically active bacterial communities involved in PAH and toxicity elimination from oil-contaminated sludge during anoxic/oxic oscillations.

The kinetics of polycyclic aromatic hydrocarbons (PAH) elimination from a contaminated sludge were determined in bioreactors under different conditions: continuously oxic, anoxic, and anoxic/oxic oscillations. The dynamics of metabolically active bacterial communities and their involvement in PAH degradation were followed by T-RFLP targeting 16S rRNA and ring hydroxylating dioxygenase (RHD) transcripts, respectively. PAH degradation was related to toxicity elimination using an aryl hydrocarbon receptor-responsive reporter cell line. Oxygen supply was identified as the main factor affecting the structure of bacterial communities and PAH removal. PAH-degrading bacterial communities were stable throughout the experiment in all conditions according to the presence of RHD transcripts, indicating that bacterial communities were well adapted to the presence of pollutants. Oxic and anoxic/oxic oscillating conditions showed similar levels of PAH removal at the end of the experiment despite several anoxic periods in oscillating conditions. These results highlight the role of dioxygenase activity after oxygen addition. Nevertheless, the higher toxicity elimination observed under oxic conditions suggests that some metabolites or other unidentified active compounds persisted under oscillating and anoxic conditions. Our results emphasize the importance of using complementary biological, chemical and toxicological approaches to implement efficient bioremediation strategies.

Second order inverse response process identification from transient step response.

Simple algorithms for identification of inverse response models from step response are difficult to obtain because analytically the solution of a system of coupled nonlinear equations is required. In this article we propose a simple identification procedure for second order inverse response processes, based on the plant step response. The algorithm provides the model parameters in a sequential way, thus avoiding the solution of a nonlinear equation system. Moreover the algorithm is flexible because it can be suited to user requirements, thus modifying the algorithm performance. Finally error bounds on the identified parameters are provided which are useful if the model is used for control design purposes.

A stable fish reporter cell line to study estrogen receptor transactivation by environmental (xeno)estrogens.

Cross-species differences between human and fish estrogen receptor (ER) binding by environmental chemicals have been reported. To study ER transactivation in a fish cellular context, we stably co-transfected the PLHC-1 fish hepatoma cell line with a rainbow trout estrogen receptor (rtER) and the luciferase reporter gene driven by an estrogen response element (ERE). This new cell model, called PELN-rtER (for PLHC-1-ERE-Luciferase-Neomycin), responded to 17beta-estradiol (E2) in a both concentration- and temperature-dependent manner, as well as to environmental ER ligands from different chemical classes: natural and synthetic estrogens, zearalenone metabolites, genistein, alkyphenoles and benzophenone derivatives. The comparison with other in vitro models, i.e. human reporter cell lines (HELN-rtER, MELN) and vitellogenin induction in primary cultures of rainbow trout hepatocytes, showed an overall higher sensitivity of the human cells for a majority of ligands, except for benzophenone derivatives which were active at similar or lower concentrations in fish cells, suggesting species-specificity for these substances. Correlation analyses suggest that the fish cell line is closer to the trout hepatocyte than to the human cell context, and could serve as a relevant mechanistic tool to study ER activation in fish hepatic cellular context.

Characterisation of bioactive compounds in infant formulas using immobilised recombinant estrogen receptor-alpha affinity columns.

In this study, the use of recombinant estrogen receptor alpha (ERalpha)-based affinity columns was reported, for the isolation and the identification of estrogenic substances present in complex matrices, focusing on bioactive compounds present in foodstuff. The capability of affinity columns to trap high, but also low-affinity radio-labelled ligands (17beta-estradiol, genistein and bisphenol A) was demonstrated. Three pooled samples of infant formulas (milk-based, hypoallergenic and soy-based formulas for infants aged 0-4 months) from a EU market basket were prepared by the CASCADE Network of Excellence. After determining the estrogenic activity of these food samples, human recombinant ERalpha ligand binding domain (LBD) based affinity columns combined with suitable analytical methods (high resolution LC-MS/MS) were used to identify the bioactive compounds present in the soy-based formula extract, namely phytoestrogens (genistein and daidzein) involved in the agonistic activity measured. Incubations of genistein with liver microsomes were carried out and the extracts analysed following the same protocol, demonstrating that hERalpha affinity columns can also be used for trapping active metabolites. This approach combining bioluminescent cell lines with this useful tool based on hERalpha-LBD affinity columns thus allowed the purification and the concentration of both known and unknown estrogenic ligands prior to investigation of their structure using LC-MS.

Relevant approach to assess performances of wastewater biosolids composting in terms of micropollutants removal.

The presence of organic pollutants in wastewater biosolids and their possible impact to the environment contribute to decrease interest for the agricultural spreading of biosolids. It is thus important to have a better overview of sewage sludge quality in terms of organic pollutant content and ecotoxicity assessment. It is also necessary to better understand the impact of biosolid composting processes on the pollutant and toxicity removal. Therefore, concentrations of oestrogens (E), nonyphenol ethoxylates (NPE), polycyclic aromatic hydrocarbons (PAH), polychlorinated biphenyls (PCB) and linear alkyl benzene sulphonates (LAS) and some of their associated toxic effects were determined at different stages of a composting process using, respectively, chemical analysis and in vitro bioassays (estrogen receptor alpha, dioxin receptor and pregnan X receptor reporter cell lines). Pollutants concentrations were higher in the final compost than in biosolid due to dry matter reduction through composting. Mass balance calculation shows a positive impact of the aerobic treatment on the removal of the most degradable pollutants. The three toxicological activities were measured in both biosolids and in the initial and final compost: oestrogenic activity increased whereas dioxin-like and pregnan X activities decreased. The difficulty in correlating chemical and toxicological results underlines the importance of combining both approaches in order to improve the assessment of the compost quality.

Estrogen and antiestrogen-dependent regulation of breast cancer cell proliferation in multicellular spheroids: Influence of cell microenvironment.

Multicellular tumor spheroids, an in vitro 3-D model that simulates malignant-cell contacts within a tumor, can be used to evaluate tumor response to therapeutic agents. We found that MELN (derived from MCF-7 cells) cells grown in 3-D as spheroids, remain highly sensitive to estradiol in terms of growth, down-regulation of ERalpha expression and ERalpha-induced transcriptional activity. Estradiol induces cyclin D1 and CDK1 proteins in Ki-67 positive proliferating cells, whereas survivin is up-regulated in both Ki-67 positive proliferative outer layer of cells and around the necrotic zone in non-proliferating cells. OH-Tam inhibits both estradiol-induced transcriptional activity and estradiol-dependent growth of MELN spheroids. Consistent with its antiproliferative effect, we observed that OH-Tam induces an important decrease in the proportion of proliferating cells, positive for Ki-67, cyclin D1 and CDK1. But, in contrast to what was expected, OH-Tam treatment resulted in a decrease in the proportion of p21 positive cells. Furthermore, despite its ability to down-regulate survivin in MELN spheroids, OH-Tam did not trigger apoptosis. Taken together, these results indicate that this model, is more relevant to an in vivo situation than monolayer cultures. It could be useful to identify new markers of the response to endocrine treatment and to investigate the effects of drugs combination.

Scientific management of Mediterranean coastal zone: a hybrid ocean forecasting system for oil spill and search and rescue operations.

The oil spill from Prestige tanker showed the importance of scientifically based protocols to minimize the impacts on the environment. In this work, we describe a new forecasting system to predict oil spill trajectories and their potential impacts on the coastal zone. The system is formed of three main interconnected modules that address different capabilities: (1) an operational circulation sub-system that includes nested models at different scales, data collection with near-real time assimilation, new tools for initialization or assimilation based on genetic algorithms and feature-oriented strategic sampling; (2) an oil spill coastal sub-system that allows simulation of the trajectories and fate of spilled oil together with evaluation of coastal zone vulnerability using environmental sensitivity indexes; (3) a risk management sub-system for decision support based on GIS technology. The system is applied to the Mediterranean Sea where surface currents are highly variable in space and time, and interactions between local, sub-basin and basin scale increase the non-linear interactions effects which need to be adequately resolved at each one of the intervening scales. Besides the Mediterranean Sea is a complex reduced scale ocean representing a real scientific and technological challenge for operational oceanography and particularly for oil spill response and search and rescue operations.

Las aguas y sedimentos del río tunecino Hamdoun presentan riesgo de disrupción endocrina / Water and sediment waste rejected in Hamdoun’sriver are major endocrine disrupting system

Diversos estudios recientes sostienen que muchos productos químicos antropogénicos, presentes en el medio ambiente, imitan la acción de hormonas endógenas. Estos disruptores endocrinos pueden originar múltiples efectos adversos en la fauna, como la feminización de peces, la pérdida de capacidad reproductiva, defectos congénitos y, a veces, pueden estar en el origen de algunos tipos de cáncer en el ser humano. La aparición de intersexos en peces de varios ríos europeos se ha atribuido a la exposición a sustancias químicas estrogénicas presentes en los efluentes de estaciones de tratamiento de aguas residuales. Para profundizar en el efecto ambiental de estos contaminantes, hemos investigado la actividad estrogénica, de receptor de hidrocarburo arílico y de receptor X de pregnano, de muestras de agua y sedimentos del río Hamdoun, tomadas aguas arriba y aguas abajo de la zona de vertidos procedentes del área industrial de la región central de Túnez. Mediante un ensayo in vitro de células cancerosas bioluminiscentes que expresan el gen de la luciferasa bajo el control de ciertos elementos con acción hormonal, hemos detectado escasa actividad estrogénica en agua y sedimentos por encima de la zona de vertidos; sin embargo, encontram os fuerte actividad es trogénica, de receptor de hidrocarburo arílico y de receptor X de pregnano en agua y s edimentos río abajo. Con experimentos de com petición demostramos que, predominantemente, las muestras de agua y de sedimentos con actividad estrogénica contienen compuestos con alta y baja afinidad con el ER " recombinante, respectivamente. Estos resultados indican que el agua del río y los sedimentos constituyen un importante sumidero y pueden ser fuente potencial de contaminantes disruptores endocrinos(AU)

In recent years, many studies supported that anthropogenic chemicals occurring in the environment have been shown to mimic the action of endogenous hormones. These endocrine-disrupting chemicals can potentially lead to a host of adverse effects on wildlife, such as the feminization of fish, the lack of reproduction success, birth defects and sometimes they can be the origin of some kind of cancers in human. The occurrence of intersex fish in a number of European rivers has been attributed to the exposure to estrogenic chemicals present in sewage treatment work effluents. To further understand the environmental effect of these contaminants, the estrogenic, aryl hydrocarbon receptor and pregnane X receptor activities of water and sediments were investigated in this study. The water and sediment samples were obtained from upstream and downstream outfalls of the Hamdoun River located in proximity of the industrial area in the centre region of Tunisia. Using an in vitro assay with bioluminescent cancer cells expressing luciferase gene under different hormonal responsive element control, we detected a much lower level of estrogenic activity in water and sediment upstream, however, we found out a strong estrogenic, aryl hydrocarbon receptor and pregnane X receptor activities in water and sediment downstream this river. By using competition experiments, we demonstrated that estrogenic activity found contained mainly compounds with a strong and lower affinitiy in water and sediment respectively with the recombinant ER ". These results suggest that the river water and sediments are a major sink and could be a potential source of endocrine-disrupting chemicals contaminants(AU)

Estrogenic activity of cosmetic components in reporter cell lines: parabens, UV screens, and musks.

In this work, the estrogenic effects of three classes of substances included in cosmetic formulations-parabens, ultraviolet (UV) screens, and musk fragrances-were studied. Their estrogenic activity was measured with the use of three reporter cell lines: HELN, HELN ERalpha, and HELN ERbeta. These three cell lines allowed for the measurement of estrogenic activity toward estrogen receptors alpha and beta (ERalpha and ERbeta, while taking nonspecific interactions into account. Eight of the 15 substances tested showed specific estrogenic activity with the following degree of potency on ERalpha butylparaben > propylparaben > homosalate = octyl-dimethyl-PABA = 4-methyl-benzylidenecamphor = octyl-methoxycinnamate > ethylparaben = galaxolide. Among these active substances, parabens activated ERalpha and ERbeta similarly, UV screens activated ERalpha moderately and had almost no effect on ERbeta, and fragrances did not activate ERbeta. Methylparaben, ethylparaben, musk moskene, celestolide, and cashmeran did not activate estrogenic responses up to 10(-5) M. Musk ketone and benzophenone-3 were not considered estrogenic at 10(-5) M.

A new mechanism of action for skin whitening agents: binding to the peroxisome proliferator-activated receptor.

Synopsis Octadecenedioic acid is known as a skin whitening agent but its activity is not mediated via a direct inhibition of tyrosinase. Based on the secondary properties of this molecule, such as its anti-inflammatory and anti-ageing effects, we postulated that octadecenedioic acid interacted with the peroxisome proliferator-activated receptor (PPAR) as this nuclear receptor also mediates these effects. Using reporter gene technology, we were indeed able to demonstrate binding of octadecenedioic acid to all three PPAR subtypes, in particular PPARgamma with an EC(50)-value of approx. 1 x 10(-6) m. Binding to PPARgamma of octadecenedioic acid or rosiglitazone, a known pharmaceutical PPARgamma agonist, led to reduced melanogenesis. Subsequently also tyrosinase mRNA (as measured by real-time polymerase chain reaction) and tyrosinase levels (as measured by Western blot) were reduced, suggesting the existence of a complete novel mechanism of skin whitening agents: binding to PPARgamma results in reduced tyrosinase mRNA expression which in turn results in less tyrosinase being formed. This in turn leads to reduced melanogenesis both in vitro and in vivo Because octadecenedioic acid binds not only to PPARgamma but also to PPARalpha and PPARdelta, other efficacies mediated via these receptors may also be expected.

Histone deacetylase inhibition and estrogen receptor alpha levels modulate the transcriptional activity of partial antiestrogens.

In this study, we have analysed the effects of histone deacetylase (HDAC) inhibition on estrogen receptor (ER) expression and on its transcriptional activity in response to antiestrogens. In several breast cancer cell lines, trichostatin A (TSA), a potent HDAC inhibitor, strongly decreases ERalpha expression in a dose-dependent manner. This repression is observed independently of the presence of ligand and also occurs in ovarian and endometrial cell lines. In addition, we show that in MCF7 cells bearing a stably transfected reporter plasmid (MELN cells), partial antiestrogens such as 4-OH-tamoxifen (OHTam), raloxifen or LY117018, switch to an agonist activity upon HDAC inhibition. This effect is blocked by the pure antiestrogen ICI182780 and exhibits a half-maximal concentration of OHTam equivalent to its affinity for ERalpha. The TSA-dependent decrease of ERalpha expression is required to induce the agonist switch of OHTam properties as it is lost in cells constitutively expressing exogenous receptors (MELN-ERalpha or ERbeta). By contrast, the transrepression activity of OHTam is abolished by TSA independently of the decrease of ERalpha expression. Interestingly, in MELN-ERalpha, ICI182780 remains inhibitory suggesting the involvement of HDAC-independent mechanisms. Finally, in the absence of TSA, transcriptional activity in response to OHTam is significantly raised in MELN cells expressing low levels of ERalpha after transfection of antisense oligonucleotides. In conclusion, inhibition of HDAC enzymatic activity and modulation of ERalpha levels tightly control the relative agonist activity of partial antiestrogens on a stably integrated reporter transgene.

Estrogenic activity in water and sediments of a French river: contribution of alkylphenols.

Alkylphenols, known to possess estrogenic activity, have been found in the aquatic environment. In this study, we focused on the contribution of alkylphenols to total estrogenic activity in sediment and water extracts of French rivers. Four sites representing rural, agricultural, urban, and industrial watersheds were studied. The concentrations of alkylphenols in water and sediment were quantified by GC/MS. Estrogen-responsive reporter cell lines (MELN) have been used for investigating estrogenic activity at these sites. These observed activities were compared with activities mediated by known concentrations of alkylphenols. In water, the concentration of alkylphenols, from 0.06 to 0.550 microg x L(-1) and from < 0.001 microg x L(-1) to 0.077 microg x L(-1) for nonylphenols and 4t-octylphenol, respectively, were too low to contribute to the observed estrogenic activity. In sediment of the industrial, rural, and urban sites, the observed estrogenic activities could be explained in great part by the alkylphenol concentrations from 0.26 to 2.87 microg x g(-1) and from 0.005 microg x g(-1) to 0.49 microg x g(-1) for nonylphenols and 4t-octylphenol, respectively. In the agricultural site, the alkylphenols (0.022 microg x g(-1) of nonylphenols) poorly contribute to the observed estrogenic activity. Other compounds, such as natural and synthetic hormones, present in water and sediments could act additively in the overall activity.

Disorders linked to insufficient androgen action in male children.

Virilization of the external genitalia in the male fetus requires testosterone and dihydrotestosterone (DHT), which is formed from testosterone by the action of the enzyme, 5alpha-reductase type 2 (5alphaR-2). Mediation of the effects of both testosterone and DHT requires a functional androgen receptor (AR) located in the cytoplasmic compartment of target cells. DHT (or testosterone) binding induces a conformational change which facilitates AR nuclear transport, phosphorylation and dimerization, ultimately regulating of the rate of transcription of androgen-dependent genes. Any event which impairs DHT formation (mutation within the 5alphaR-2 gene or 5alphaR-2 inhibitors) or normal function of the AR (mutation in the AR gene, antiandrogens) may result in insufficient androgen action in the male fetus and in subsequent undervirilization in the newborn. Hypospadias may be due to a defect in androgen action due to mutation of the 5alphaR-2 or of the AR gene. Mutation of unidentified genes is likely to underlie this displacement of the urethral meatus from the tip to the ventral side of the phallus. An aetiological role for environmental chemical products has been postulated, since ethnic as well as geographical differences in the incidence of hypospadias have been noted. Increasing evidence has been gathered indicating that widely used industrial and agricultural chemicals have deleterious effects on normal male sexual differentiation. Cryptorchidism and micropenis may represent an intersex phenotype, even if they are isolated. Aetiological factors include 5alphaR-2 gene mutation, AR gene mutation or environmental hormonal disruptors. In conclusion, several phenotypes have been attributed to insufficient androgen action during fetal life. Whereas mutations in the 5alphaR-2 gene and AR gene are natural, attention should be focused on environmental endocrine disruptors that are able to mimic steroid 5alpha-reductase deficiency or partial androgen insensitivity syndrome.

Environmental xenoestrogens, antiandrogens and disorders of male sexual differentiation.

Over the past 20 years, the documented increase in the disorders of male sexual differentiation, such as hypospadias, cryptorchidism, and micropenis, has led to the suspicion that environmental chemicals are detrimental to normal male genital development in utero. Male sexual differentiation is critically dependent on the normal action of androgens, and unbalanced androgen/estrogen ratios can disturb it. Environmental xenoestrogens (such as herbicides, pesticides, PCBs, plasticizers, and polystyrenes) that mimic estrogens or environmental antiandrogens (such as polyaromatic hydrocarbons, linuron, vinclozolin, and pp'DDE) that disturb endocrine balance, cause demasculinizing effects in the male foetus. These environmental chemicals are often referred to as endocrine disruptors: they are thought to mimic endogenous estrogens by entering the cell, binding to the receptor and activating transcription, they may also antagonize normal androgen action. We have established numerous cell lines to assess the estrogenicity and antiandrogenicity of compounds found in the environment and to identify new products present in wastewater effluents that are able to disrupt endocrine functions. Several cell lines responding to estrogens have been obtained in our group, including cells with different enzymatic equipment and cells expressing chimeric receptor or natural estrogen receptors alpha and beta. These cell lines have proved to be useful for assessing the biological activity of pesticides, fungicides, and chemicals found in plastic or discarded in the environment. In order to generate a powerful tool for the investigation of androgen action and the rapid screening of potential antagonists, we developed a new stable prostatic cell line. The PALM cell line is an original cellular model to characterize the response of hAR, and it provides an easy and rapid bioluminescent test to identify new antagonists. We also developed a model based on a fusion protein between the androgen receptor (AR) and the green fluorescent protein (GFP) to study the intracellular dynamics of AR. The GFP-AR model was applied to define the ability of several xenoestrogens and antiandrogens to inhibit the nuclear transfer of AR. The ubiquitous presence of endocrine disruptors in the environment and the increased incidence of neonatal genital malformation support the hypothesis that disturbed male sexual differentiation may in some cases be caused by increased exposure to environmental xenoestrogens and/or antiandrogens.

Reporter cell lines are useful tools for monitoring biological activity of nuclear receptor ligands.

To characterize the specificity of synthetic compounds for nuclear receptors, we established stable cell lines expressing the luciferase gene and different wild-type or chimaeric receptors. MCF-7 cells, which express the oestrogen receptor alpha (ER alpha), and HeLa cells, which do not express the oestrogen receptor, were transfected with a plasmid containing the luciferase gene downstream from a minimum promoter (beta-globin) and an oestrogen-responsive element, generating the MELN and the HELN cell lines, respectively. MELN cells enabled the detection of compounds that bind to the ER alpha or interfere with its pathway. HELN cells were used to establish stable transfectants expressing different nuclear receptors containing the DNA-binding domain of the oestrogen receptors. We thus established ER alpha or ER beta reporter cell lines by transfecting ER alpha or ER beta expression plasmids, and also retinoic acid receptor alpha, beta or gamma reporter cell lines by transfecting the chimaeric RAR gene, in which the DNA-binding domain was replaced by the ER alpha DNA-binding domain.

Correlation between different gene expression assays designed to measure trans-activation potencies of systemic glucocorticoids.

The glucocorticoids (GC) betamethasone, dexamethasone, hydrocortisone, methylprednisolone, prednisolone and triamcinolone acetonide are currently used in the treatment of inflammatory diseases. Through a process called trans-activation, GC activate gene expression and produce various physiological and pharmacological effects. In particular, by inducing gluconeogenic enzymes, long-term GC treatment may cause diabetes. Using three different assays, we have extensively compared the capacity of the above GC to activate gene expression. trans-Activation of a GC inducible luciferase gene was assessed in HeLa and A549 cells after stable and transient transfection, respectively. In hepatoma tissue culture cells, we measured trans-activation of the endogenous gene encoding tyrosine aminotransferase, a gluconeogenic enzyme. Half-maximal effective concentrations of GC were determined by dose-response analyses. Results obtained with these assays were highly correlated and GC were ranked in three groups according to their trans-activation potency: betamethasone, dexamethasone, and triamcinolone acetonide > methylprednisolone and prednisolone > hydrocortisone. Potencies were not strictly related to receptor binding affinities and not significantly affected by the amount of endogenous GC receptor.

Transcriptional potencies of inhaled glucocorticoids.

Glucocorticoids (GC) are the most effective anti-inflammatory drugs used in asthma. By a process called trans-activation, they increase the transcription of genes involved in either beneficial processes or certain side effects. Through trans-repression, they inhibit the transcription factors nuclear factor kappa B (NF-kappaB) and activator protein-1 (AP-1), thereby decreasing the expression of many genes encoding inflammatory mediators such as the cytokine RANTES. We have measured the trans-activation and trans-repression potencies of the five currently available inhaled GC using reporter gene assays. The rank order of trans-activation potencies in HeLa cells stably transfected with a GC-inducible luciferase gene was fluticasone propionate > budesonide and triamcinolone acetonide > beclomethasone dipropionate and flunisolide. For all GC except beclomethasone dipropionate, there was a highly significant correlation between their potency to trans-activate in HeLa cells and their capacity to induce the gluconeogenic enzyme tyrosine aminotransferase in hepatoma tissue culture (HTC) cells. The rank order of trans-repression potencies in A549 lung cells transiently transfected with an AP-1- or NF-kappaB-dependent luciferase gene was fluticasone propionate > budesonide > beclomethasone dipropionate, triamcinolone acetonide, and flunisolide. The same rank order was found for inhibition of RANTES release. Thus, determination of trans-repression and trans-activation potencies of GC may help to predict their capacity to produce anti-inflammatory and side effects, respectively.

A stable prostatic bioluminescent cell line to investigate androgen and antiandrogen effects.

We developed a new stable prostatic cell line expressing the human androgen receptor (AR) and the AR-responsive reporter gene to generate a powerful tool for investigating androgen action and for rapid screening of agonists and antagonists. The AR-deficient PC-3 cells were stably transfected with pSG(5)-puro-hAR and pMMTV-neo-Luc. After selection with puromycin and neomycin, one highly inducible clone was isolated and named PALM, for PC-3-Androgen receptor-Luciferase-MMTV. The expression of hAR was confirmed by western blot and steroid-binding assays on the whole cells. The transcriptional activity of the clone was measured after incubation of cells with increasing concentrations of synthetic R1881 or natural androgens (DHT and testosterone). The three agonists had the same maximal activity at 0.1 microM and the fold induction was equal to 20. The agonist and antagonist activities of the steroidal antiandrogens (cyproterone acetate and RU2956) and the non-steroidal antiandrogens (nilutamide, bicalutamide, inocoterone and hydroxyflutamide) measured with the PALM cells were in good correlation with the results obtained with transiently transfected cells. The selectivity in steroid transactivation was demonstrated with estradiol, progesterone, cortisol, dexamethasone and aldosterone. Spironolactone and RU486 showed partial agonist and antagonist activities, whereas R5020 presented only a partial antagonist activity. We here demonstrate that this stable transfectant provides an accurate tool for studying wild-type human AR activation and its regulation by androgens and antiandrogens in a human prostatic epithelial cell, which is routinely available and remains androgen-responsive in vitro.

Reporter cell lines to study the estrogenic effects of xenoestrogens.

In order to characterize the estrogenic activity of chemicals, we established complementary in vitro recombinant receptor-reporter gene assays in stably transfected MCF-7 and HeLa cells. MCF-7 cells which express the endogenous estrogen receptor alpha (ER alpha) were stably transfected with only an estrogen-regulated luciferase gene. These cells enable the detection of compounds which bind to ER alpha or interfere with the induction of ER alpha mediated gene expression. Furthermore, HeLa cells, which do not express endogenous ERs, were transfected with an ER alpha or an ER beta construct together with an estrogen-regulated luciferase gene, or a chimeric GAL4-ER alpha receptor and the corresponding luciferase reporter gene. Finally, we tested these four cellular models as tools to check the estrogenic activities of several potential xenoestrogens and to detect estrogenic activity in wastewater sewage treatment effluents. In all of the models, nonylphenol mixture (NPm), 4n-nonylphenol (4nNP), 2,4'-DDE, 4,4'-DDE and wastewater sewage treatment effluent were active, while PCB mixture (Aroclor 1254), PCB 77, atrazine and lindane (gamma hexachlorocyclohexane) were inactive. Dioxin partially activates the estrogen receptor in MCF-7 cells while in HeLa-derived cell lines, it decreased the estrogenic-induced expression of luciferase.