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.

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.

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.

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.

Antiestrogenic effects of all-trans-retinoic acid and 1,25-dihydroxyvitamin D3 in breast cancer cells occur at the estrogen response element level but through different molecular mechanisms.

Most breast tumors show estrogen-dependent growth and are thus susceptible to antiestrogenic therapy. MCF-7 cells, obtained from a human estrogen-dependent breast carcinoma, are widely used for studying the modulation of estrogenic responses by different effectors. All-trans-retinoic acid (RA) and 1,25-dihydroxyvitamin D3 (Vit D3) inhibited estrogen-induced growth of MCF-7 cells and their effect was potentiated by the classical antiestrogen, hydroxytamoxifen. In MCF-7 cells, we found that RA and Vit D3 also inhibited estrogen-induced transcription; this was shown both for an endogenous gene (pS2) and for various exogenous transfected genes. Their inhibitory effect could not be reversed by increasing estradiol concentrations, showing that contrary to classical antiestrogens, they did not compete with estradiol to bind the estrogen receptor (ER). Analysis of the inhibitory mechanisms indicates that RA and Vit D3 receptors can directly or indirectly impair the binding of ER to the estrogen responsive element. The antagonist effect of RA would be found especially at DNA level since it seems to essentially involve an estrogen responsive element. The antagonist effect of Vit D3 would be found especially at the ER level since it seems to concern estrogen binding and dimerization domains of ER. We conclude that the antiestrogenic effects of RA and Vit D3 are similar since they can, via their receptors, interfere with estrogenic action at the estrogen responsive element level but that they are not identical since different molecular mechanisms are involved.

Differential expression of the RTP/Drg1/Ndr1 gene product in proliferating and growth arrested cells.

Using a differential display method to identify differentiation-related genes in human myelomonocytic U937 cells, we cloned the cDNA of a gene identical to Drg1 and homologous to other recently discovered genes, respectively human RTP and Cap43 and mouse Ndr1 and TDD5 genes. Their open reading frames encode proteins highly conserved between mouse and man but which do not share homology with other know proteins. Conditions in which mRNAs are up-regulated suggest a role for the protein in cell growth arrest and terminal differentiation. We raised antibodies against a synthetic peptide reproducing a characteristic sequence of the putative polypeptide chain. These antibodies revealed a protein with the expected 43 kDa molecular mass, up-regulated by phorbol ester, retinoids and 1,25-(OH)2 vitamin D3 in U937 cells. It was increased in mammary carcinoma MCF-7 cells treated by retinoids and by the anti-estrogen ICI 182,780 but not by 4-hydroxytamoxifen. The mouse Drg1 homologous protein was up-regulated by retinoic acid in C2 myogenic cells. The diversity of situations in which expression of RTP/Drg1/Ndr1 has now been observed shows that it is widely distributed and up-regulated by various agents. Here we show that ligands of nuclear transcription factors involved in cell differentiation are among the inducers of this novel protein.

RIP 140 enhances nuclear receptor-dependent transcription in vivo in yeast.

RIP140 has previously been cloned as a factor that interacts with the estrogen receptor (ER) in vitro. We demonstrate in this study that RIP140 is a co-factor for nuclear receptor in yeast. RIP140 enhances the ER transcriptional activity by increasing 1.5- to 4-fold the induction factor of the reporter gene response at saturating hormone concentrations, this effect being magnified at suboptimal doses of estradiol. Moreover, RIP140 decreases the ED50 of the dose-response curve. These effects are recovered with an N-terminal truncated ER, but impaired by point mutations that abolish AF2-AD activity. We did not observe any modulation of the partial agonist 4-hydroxytamoxifen activity in the presence of RIP140. Thus, RIP140 modulates transcriptional activity of ER through the AF2-AD domain and in a agonist-dependent fashion. RIP140 is also a strong coactivator for the retinoid pathway, as its expression enhances 10-fold the transactivation of a chimeric retinoic acid-alpha receptor at saturant hormone concentration and left shifted 5-fold the ED50 of the dose-response curve. We have investigated whether RIP140 could be involved in cross-talk between estrogenic and retinoid pathways.

Mutation of isoleucine 747 by a threonine alters the ligand responsiveness of the human glucocorticoid receptor.

Mutation of isoleucine 747 to threonine in the C-terminal part of the ligand-binding domain (LBD) of the human glucocorticoid receptor (GR) alters the ligand specificity for transactivation. Natural glucocorticoids such as cortisol or corticosterone were completely inactive with the mutant 1747T, whereas synthetic steroids like dexamethasone efficiently stimulated GR 1747T-mediated transactivation. However, the corresponding ligand dose-response curve for dexamethasone-induced transactivation was shifted to higher concentrations when compared with that obtained with the wild type GR. Neither this shift nor the inability of cortisol to activate the 1747T mutant was due to an altered in vitro ligand-binding affinity. In the canonical three-dimensional structure of nuclear receptor LBDs, isoleucine 747 is in the direct vicinity of residues that contribute to the ligand-binding pocket. Moreover, it is located in the C-terminal LBD region, which harbors the conserved core of the activation function AF-2 and undergoes a ligand-induce transconformation, required to generate the surface interacting with putative transcriptional intermediary factors/coactivators of AF-2. The phenotype of 1747T mutant is discussed in view of the possible consequences of the mutation on the various events which, according to the model, lead to a transcriptionally competent AF-2.

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.

A multiple luminescent procedure for the detection of different papillomaviruses on dot blots.

We developed a method based on the use of various luminescent systems for identification of several nucleic acid sequences on the same dot blots. In a simultaneous assay, the target DNA molecules were hybridized with different DNA probes. These probes were labelled with biotin or digoxigenin or directly coupled to enzymes such as glucose-6-phosphate dehydrogenase, peroxidase or alkaline phosphatase. After hybridization, these labels were detected by luminescent reactions using an amplified camera. Rapid detection and specific identification of pathogenic agents such as human papillomaviruses (HPV) could be performed in a single step by this procedure. Polymerase chain reaction was carried out using general primers and virus types were identified on dot blots using short HPV 11, 16 and 18 specific oligonucleotides.

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.

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.

Development of specific bioluminescent in vitro assays for selecting potential antimineralocorticoids.

Efficient antimineralocorticoid selection requires a reliable, discriminating and easy assay for monitoring biological activity of not only the specific receptor, but also closely related receptors such as glucocorticoid and progestin. These related activities should be as low as possible to obtain specific antimineralocorticoid compounds. In this paper, we describe two cellular models used for easy and specific measurement of mineralocorticoid and progestin activities. These models involve the induction of firefly luciferase under hormonal control mediated by a chimeric receptor. The first model comprises transiently transfected MCF-7 cells, whereas the second uses stably transfected HeLa cells. Glucocorticoid activity was assayed with the classic tyrosine-aminotransferase induction method in HTC cells. Six compounds of a new family of 11 beta-substituted-17-spirolactone steroids were thus studied and compared to control compounds. Five of them showed antimineralocorticoid activity and one was active at a concentration lower than that of mespirenone.

In vitro and in vivo interactions between nuclear receptors at estrogen response elements.

To study mechanisms involved in the antiestrogenic effect of retinoic acid (RA), previously described in mammalian cells, we used in vitro and in vivo approaches. One hypothesis was direct competition between nuclear receptors (ER, RAR and RXR) at the DNA level. We first showed in vitro that the RAR/RXR heterodimer could weakly bind an ERE and that retinoid receptors reduced binding of ER to an ERE. We next checked whether, in yeast, direct competition between receptors that recognize the same responsive element could be monitored in a reconstituted heterologous estrogen-responsive system, by determining the expression of a reporter gene. We then co-transformed RAR and RXR in an estrogenic responsive strain. This model demonstrated that, even though RAR/RXR was able to bind an ERE, the addition of retinoic acid had no inhibitory effect on estrogen-induced responses in this yeast system, unlike in mammalian cells. Interference between these receptors should require other factors than interactions at the ERE level. This model could be used to identify mammalian factors interacting with estrogen and retinoic acid receptors which could play a role in crosstalk between these receptors.

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.

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.

[Digestive hemorrhage secondary to ileocecal actinomycosis]. / Hemorragia digestiva secundaria a actinomicosis ileocecal.

Report of a case of abdominal actinomycosis, including the cecoapendicular region, that clinically presented as lower digestive bleeding, and was diagnosed by the anatomopathological study of the surgical resection specimen, since the patient was operated with the preoperative diagnosis of vascular malformation. This type of presentation is uncommon in abdominal actinomycosis, and explains the presentation of this clinical case.