Elena Gomez

Binding of estrogenic compounds to recombinant estrogen receptor-alpha: application to environmental analysis.

Estrogenic activity in environmental samples could be mediated through a wide variety of compounds and by various mechanisms. High-affinity compounds for estrogen receptors (ERs), such as natural or synthetic estrogens, as well as low-affinity compounds such as alkylphenols, phthalates, and polychlorinated biphenyls are present in water and sediment samples. Furthermore, compounds such as polycyclic aromatic hydrocarbons, which do not bind ERs, modulate estrogen activity by means of the aryl hydrocarbon receptor (AhR). In order to characterize compounds that mediate estrogenic activity in river water and sediment samples, we developed a tool based on the ER-αligand-binding domain, which permitted us to estimate contaminating estrogenic compound affinities. We designed a simple transactivation assay in which compounds of high affinity were captured by limited amounts of recombinant ER-αand whose capture led to a selective inhibition of transactivation. This approach allowed us to bring to light that water samples contain estrogenic compounds that display a high affinity for ERs but are present at low concentrations. In sediment samples, on the contrary, we showed that estrogenic compounds possess a low affinity and are present at high concentration. Finally, we used immobilized recombinant ER-αto separate ligands for ER and AhR that are present in river sediments. Immobilized ER-α, which does not retain dioxin-like compounds, enabled us to isolate and concentrate ER ligands to facilitate their further analysis.

Distribution of chromium species in a Cr-polluted soil: Presence of Cr(III) in glomalin related protein fraction

The accumulation of Cr in soil could be highly toxic to human health; therefore Cr soil distribution was studied in rhizosphere soils from Ricinus communis and Conium maculatum and bare soil (BS) from an industrial and urban area in Argentina. Total Cr, Cr(VI) and Cr(III) concentrations were determined in 3 soil fractions: total, extractable and associated to total-glomalin-related protein (T-GRSP). BS had the highest total Cr and total Cr(VI) concentrations. Total Cr(VI) concentration from both rhizosphere soils did not differ from the allowed value for residential area in Argentina (8 μg Cr(VI) g(-1) soil), while total Cr(VI) in BS was 1.8 times higher. Total Cr concentration in all the soils was higher than the allowed value (250 μg Cr g(-1) soil). Extractable and associated to T-GRSP Cr(VI) concentrations were below the detection limit. Cr(III) bound to T-GRSP was the highest in the BS. These findings are in agreement with a long term effect of glomalin in sequestrating Cr. In both plant species, total Cr was higher in root than in shoot and both species presented arbuscular mycorrhizal fungi (AMF). As far as we know, this is the first study that reports the presence of Cr in T-GRSP fraction of soil organic matter. These findings suggest that Cr mycorrhizostabilization could be a predominant mechanism used by R. communis and C. maculatum to diminish Cr soil concentration. Nevertheless, further research is needed to clarify the contribution of native AMF isolated from R. communis and C. maculatum rhizosphere to the Cr phytoremediation process.

Reporter Cell Lines to Monitor Steroid and Antisteroid Potential of Environmental Samples

In order to monitor the (anti)steroid activity of environmental samples, we established stable cell lines expressing luciferase under the control of estrogens, androgens, progestives and glucocorticoids. The breast cancer MCF-7 cells which express the estrogen receptor α (ERα), the glucocorticoid receptor (GR) and the progesterone receptor (PR) were transfected by an estrogen (ERE-βGlob-Luc) or a glucocorticoid/progestin/androgen (MMTV-Luc) regulated luciferase plasmid in order to enable the detection of compounds which bind both ERα, PR and GR (MELN and MMLN cells). Human prostatic cells PC3 were stably transfected by both an androgen receptor gene and the MMTV-luciferase plasmid (PALM cells). These three cellular models were validated as tools to check the estrogenic, progestive, glucocorticoid and androgenic activities using several potential xenohormones and environmental samples. As these environmental samples were fractionated after solid phase extraction to isolate active compounds, we used these cellular models to monitor the different fractions. In the estrogenic model mid-polar fractions of environmental samples were found active while in the androgenic model, the same fractions had antagonist activity.

Ecotoxicological assessment of soil microbial community tolerance to glyphosate

Glyphosate is the most used herbicide worldwide. While contrasting results have been observed related with its impact on soil microbial communities, more studies are necessary to elucidate the potential effects of the herbicide. Differences in tolerance detected by Pollution Induced Community Tolerance (PICT) approach could reflect these effects. The objective of the present study was to assess the tolerance to glyphosate (the active ingredient and a commercial formulation) of contrasting soils with (H) and without (NH) history of exposure. The hypothesis of a higher tolerance in H soils due to a sustained selection pressure on community structure was tested through the PICT approach. Results indicated that tolerance to glyphosate is not consistent with previous history of exposure to the herbicide either for the active ingredient or for a commercial formulation. Soils of H and NH sites were also characterized in order to determine to what extent they differ in their functional diversity and structure of microbial communities. Denaturant Gradient Gel Electrophoresis (DGGE) and Quantitative Real Time PCR (Q-PCR) indicated high similarity of Eubacteria profiles as well as no significant differences in abundance, respectively, between H and NH sites. Community level physiological profiling (CLPP) indicated some differences in respiration of specific sources but functional diversity was very similar as reflected by catabolic evenness (E). These results support PICT assay, which ideally requires soils with differences in their exposure to the contaminant but minor differences in other characteristics. This is, to our knowledge, the first report of PICT approach with glyphosate examining tolerance at soil microbial community level.

Monitoring Endocrine Disrupter Compounds in the Tunisian Hamdoun River using In Vitro Bioassays

Anthropogenic chemicals occurring in the environment, namely endocrine-disrupting chemicals (EDCs), have generated growing concern over their potential adverse effects on human wildlife health and ecosystem processes. This interest resulted particularly from their abilities to mimic the effect of endogenous hormones. In this study, we used stable transfected reporter cell lines to investigate the endocrine-disrupting profile of water as well as sediment samples. Samples are collected from up- and downstream of an industrial wastewater discharge point at the Hamdoun River in the vicinity of an industrial zone located at the center of Tunisia. The analysis of estrogen, androgen, and xenobiotic (pregnane X and dioxin) ligands receptors expressed by chimeric cell lines indicated that while the water and sediment samples from upstream sites have lower levels of estrogenic activity, those from downstream exhibited stronger estrogenic, aryl hydrocarbon receptor (AhR), and Pregnane X Receptor (PXR) activities. Moreover, collected samples have shown hormonal activity in terms of all tested receptors except the androgenic ones. In vitro recombinant estrogen receptor competitive binding assays revealed that while the estrogenic activities of the downstream water sample compounds had a strong affinity for estrogen receptor α (ERα), those present in the sediment samples showed a weaker one. These findings were consolidated by subsequent chemical analysis (high-performance liquid chromatography with UV detectors). Our results indicate that the water and sediment discharges at the Hamdoun River represent a major sink for EDCs from natural and industrial effluents, particularly those of the textile industry, with pernicious potential to disrupt normal endocrine functions.

Accumulation des sels nutritifs dans un sédiment lagunaire et environnement hydrodynamique

Abstract The Mejean-Perols Lagoon is situated on the French Mediterranean coast. It is a small (747 ha) and shallow (70 cm) lagoon. Some treated effluent from Montpellier flows into this hypereutrophic lagoon. The cartography of C, N and P concentrations in the sediment was studied based on 35 bottom samples. The nitrogen and organic carbon concentrations were more or less homogeneous, the highest concentrations being found in the middle of the lagoon. The phosphate was principally bound onto the sediment in the west zone. The observed distribution was independent of grain size, the distribution of which was rather homogeneous. In this lagoon water circulation is wind induced. Four different water flow structures were observed, as functions of the prevailing winds (NE, NW, SE. SW). The /ones with the highest values of hydrodynamic variables, such as the velocity fields, corresponded to the highest mineral carbon and total phosphate concentrations and with lower organic carbon and nitrogen concentrations. Accumulation of organic carbon and organic nitrogen was related to the sedimentation of macroalgae biomass. The distribution of these two elements was dependent on the hydrodynamics, which stimulated their mineralization in the zones of turbulent water and their accumulation in the quieter ones. Phosphate distribution depended also on the external input from the watershed. Phosphate bound onto the sediment accumulated in the western part of the lagoon, as consequence of wind induced resuspension. It was principally present in inorganic form (70 % of total P). The concentration of phosphate extracted with Na-EDTA (calcium-bound) was higher than that of the phosphate extracted with Ca-NTA (iron-bound). The organic fractions represented 29 % and dissolved phosphate 1 % of the total P. The phosphate mobility accounted for 30 % of total phosphate.

Temporal study of estrogenic responses of mussel (Mytilus galloprovinciallis) extracts applied to reporter cell lines

In vitro bioassays would facilitate monitoring of estrogen-like compounds in mussels (Mytilus galloprovincialis) since they tend to accumulate lipophilic compounds in their fat and muscle tissue. However, estradiol (E2) steroid (already identified in mussels) could induce false positive responses. This study focuses on temporal variability in estrogenic responses of mussel extracts and the possible relation of this response with E2 levels. The reproductive cycle and growth were thus monitored for six months in sexed mussels. E2 levels were similar between genders and these levels varied with reproductive stages. human estrogen receptor (hER) activities were similar at all stages except in February. E2 present in male and female extracts accounted for part of the hER activation observed, but no correlation was found between E2 levels and hER activities.

Soil ecotoxicity assessment of glyphosate use under field conditions: microbial activity and community structure of Eubacteria and ammonia-oxidising bacteria

BACKGROUND A plot-scale experiment was conducted to assess the impact of field application rates of glyphosate on soil microbial communities by taking measurements of microbial activity (in terms of substrate-induced respiration and enzyme activity) in parallel with culture-independent approaches to assessing both bacterial abundance and diversity. Two rates of glyphosate, alone or in a mixture with 2,4-dichlorophenoxyacetic acid, were applied directly onto the soil surface, simulating normal use in chemical fallow in no-till systems. RESULTS No consistent rate-dependent responses were observed in the microbial activity parameters investigated in the field plots that were exposed to glyphosate. Denaturant gradient gel electrophoresis (DGGE) of the overall bacterial community (Eubacteria) and ammonia-oxidising bacteria (AOB) revealed no effects of the high rate of glyphosate on the structure of the communities in comparison with the control. No treatment effects were observed on the abundance of Eubacteria shortly after treatment in 2010, while a small but significant difference between the high rate and the control was detected in the first sampling in 2011. The abundance of AOB was relatively low during the study, and treatment effects were undetectable. CONCLUSIONS The absence of negative effects on soil microbial communities in this study suggests that glyphosate use at recommended rates poses low risk to the microbiota.

Soil Microbial Community-Level Physiological Profiling as Related to Carbon and Nitrogen Availability Under Different Land Uses

Abstract The goal of this work was to assess soil microbial respiration, determined by the assay of community-level physiological profiling in an oxygen-sensitive microplate (O 2 -CLPP), in response to endogenous C and several individual C substrates in the soils with different organic C contents (as a function of soil type and management practice). We also used the O 2 -CLPP to determine the respiratory response of these soils to endogenous C and amended C substrates with N addition. A respiratory quotient (RQ) was calculated based on the ratio of the response to endogenous soil C vs. each C-only substrate, and was related to total organic carbon (TOC). For assessing N availability for microbial activity, the effect of N supplementation on soil respiration, expressed as N ratio , was calculated based on the response of several substrates to N addition relative to the response without N. Soils clustered in 4 groups after a principal component analysis (PCA), based on TOC and their respiratory responses to substrates and endogenous C. These groups reflected differences among soils in their geographic origin, land use and C content. Calculated RQ values were significantly lower in natural forest soils than in managed soils for most C-only substrates. TOC was negatively correlated with RQ ( r = −0.65), indicating that the soils with higher organic matter content increased respiratory efficiency. The N addition in the assay in the absence of C amendment (i.e., only endogenous soil C present) had no effect on microbial respiration in any soil, indicating that these soils were not intrinsically N-limited, but substrate-dependent variation in N ratio within soil groups was observed.