The Dicentrarchus labrax estrogen screen test: a relevant tool to screen estrogen-like endocrine disrupting chemicals in the aquatic environment.

In response to the need for the diversification of regulatory bioassays to screen estrogen-like endocrine disrupting chemical (EEDC) in the environment, we propose the use of a reporter gene assay involving all nuclear estrogen receptors from Dicentrarchus labrax (i.e., sbEsr1, sbEsr2a, or sbEsr2b). Named DLES test (D. labrax estrogen screen), it aims at complementing existing standardized in vitro tests by implementing more estrogen receptors notably those that do not originate from humans. Positive responses were obtained with all three estrogen receptors, and-consistently with observations from other species-variations in sensitivity to E2 were measured. Sensitivity and EC50 values could be classified as follows: sbEsr2b < sbEsr2a < sbEsr1. The pharmacological characterization with a human estrogen receptor antagonist (fulvestrant) successfully validated the specific involvement of each sbEsr and evidenced the capacity of the DLES test to highlight antagonist interactions. The DLES test was applied to WWTP contaminant extracts. A positive response was detected in the inflow sample in accordance with the YES test, but not in the outflow sample. Notwithstanding, the DLES test (sbEsr2b) exhibited greater sensitivity for the screening of those samples. This study demonstrates the need for more comprehensive testing including representatives of marine species for a better detection of EEDCs. The DLES test appears as a pertinent tool to predict adverse effects and to widen the scope of screening and hazard assessment of EEDCs in the environment.

Relevance of flounder caging and proteomics to explore the impact of a major industrial accident caused by fire on the Seine estuarine water quality.

On September 26th 2019, a major fire occurred in the Lubrizol factory located near the Seine estuary, in Rouen-France. Juvenile flounders were captured in the Canche estuary (a reference system) and caged one month in the Canche and in the Seine downstream the accident site. No significant increases of PAHs, PCBs and PFAS was detected in Seine vs Canche sediments after the accident, but a significant increase of dioxins and furans was observed in water and sewage sludge in the Rouen wastewater treatment plant. The proteomics approach highlighted a dysregulation of proteins associated with cholesterol synthesis and lipid metabolism, in fish caged in the Seine. The overall results suggested that the fire produced air borne dioxins and furans that got deposited on soil and subsequently entered in the Seine estuarine waters via runoff; thus contaminating fish preys and caged flounders in the Seine estuary.

Integration of environmental signatures and omics-based approaches on the European flounder to assist with health assessment of estuarine ecosystems in Brittany, France.

This study aimed to develop a multidisciplinary approach to assess the ecological status of six moderate-sized French estuaries. For each estuary, we gathered geographical information, hydrobiological data, chemistry of pollutants and fish biology, including integration of proteomics and transcriptomics data. This integrative study covered the entire hydrological system studied, from the watershed to the estuary, and considered all the anthropogenic factors that can impact this environment. To reach this goal, European flounder (Platichthys flesus) were collected from six estuaries in September, which ensures a minimum residence time of five months within an estuary. Geographical metrics are used to characterize land use in each watershed. The concentrations of nitrite, nitrate, organic pollutants, and trace elements were measured in water, sediments and biota. All of these environmental parameters allowed to set up a typology of estuaries. Classical fish biomarkers, coupled with molecular data from transcriptomics and shotgun proteomics, highlighted the flounder's responses to stressors in its environment. We analysed the protein abundances and gene expression levels in the liver of fish from the different estuaries. We showed clear positive deregulation of proteins associated with xenobiotic detoxification in a system characterized by a large population density and industrial activity, as well as in a predominantly agricultural catchment area (mostly cultures of vegetables and pig breeding) mainly impacted by pesticides. Fish from the latter estuary also displayed strong deregulation of the urea cycle, most probably related to high nitrogen load. Proteomic and transcriptomic data also revealed a deregulation of proteins and genes related to the response to hypoxia, and a probable endocrine disruption in some estuaries. Coupling these data allowed the precise identification of the main stressors interacting within each hydrosystem.

Bioaccumulation of trace metal elements and biomarker responses in caged juvenile flounder at a polluted site: Effects of fish density and time exposure.

This study investigates the effect of fish density and exposure duration on trace metal elements (TME) bioaccumulation and several biomarkers response. Juvenile flounders were caged at low, medium and high densities and exposed during 15 or 30 days in the Seine estuary. The concentrations of the TME measured in the muscle of the caged fish were all in agreement with their bioavailability percentage in the sediments. Higher concentrations of TME were found in flounders' muscle exposed for 15 days compared with those caged for 30 days. For the same exposure time, the density of fish had no effect on the accumulation of the TME in the flounders' muscle. Biomarkers responses varied according to density and duration of exposure. Special care should be taken in their interpretation. We underline that for an optimal assessment of TME pollution in the field, 15 days with low densities of fish per cage are sufficient.

Effects of 17α-Ethinylestradiol (EE2) exposure during early life development on the gonadotropic axis ontogenesis of the European sea bass, Dicentrarchus labrax.

Exposure of young organisms to oestrogenic endocrine disrupting chemicals (EDCs) can elicit adverse effects, particularly on the reproductive function. In fish, as in other vertebrates, reproduction is controlled by the neuroendocrine gonadotropic axis, whose components are mainly regulated by sex steroids and may then be targets for EDCs. In the present study, we investigated the effects of a xenoestrogen exposure on the ontogenesis of the gonadotropic axis in European sea bass. After exposure of hatching larvae for 8 days to 17α-ethinylestradiol (EE2) (0.5 nM and 50 nM), gene expression for kisspeptins (kiss1, kiss2), gonadotropin-releasing hormones (gnrh1, gnrh2, gnrh3), gonadotropin beta subunits (lhß and fshß) and brain type aromatase (cyp19a1b) were measured using quantitative real-time PCR. Our results demonstrate that EE2 strongly stimulated the expression of brain type aromatase (cyp19a1b) in sea bass larvae. In addition, EE2 exposure also affected the mRNA levels of kiss1, gnrh1 and gnrh3 by inducing a downregulation of these genes during the early developmental stages, while no effect was seen in gnrh2, lhß and fshß. These results reinforce the idea that the larval development is a sensitive critical period in regard to endocrine disruption and that the gonadotropic axis in the developing sea bass is sensitive to xenoestrogen exposure.

Impacts of chemical stress, season, and climate change on the flounder population of the highly anthropised Seine estuary (France).

The main objective of this study was to improve our knowledge on the responses of fish populations to multistress (diffuse pollution and warming waters) in estuaries. Adult flounders were caught in two estuaries in the Eastern English Channel: the heavily polluted Seine estuary vs the moderately contaminated Canche estuary. Fish samplings were conducted in January just before the reproduction period, and in July when gonads were at rest. The overall rise in coastal winter water temperatures detected over the Channel impairs the flounder's phenology of reproduction in the two estuaries, inducing a delay of maturation process and probably also spawning. The higher liver histopathology index in Seine vs Canche could be the consequence of the fish exposition to a complex cocktail of contaminants in a strongly industrialized estuary. Higher levels of neurotoxicity, gill lipid peroxidation, and liver EROD activity were observed in Seine vs Canche. Furthermore, a possible impairment in mitochondrial metabolism was suggested in the Seine flounder population. We confirmed in this study the potential role of two membrane lipids (sphingomyelin and phosphatidylserine) in the resistance towards oxidative stress in Seine and Canche. Finally, we suggest that the Seine flounder population (and possibly the connected Eastern English Channel flounder populations over the French Coast) could be seriously impacted in the future by multistress: higher winter temperatures and chemical contamination.

Differences in chemical contaminants bioaccumulation and ecotoxicology biomarkers in Mytilus edulis and Mytilus galloprovincialis and their hybrids.

The Mytilus mussels are spread all over the world and many related species coexist in several areas and can produce hybrid offspring. Mussels have been used for decades in national and international programs to monitor chemical contamination in the environment. Differences in bioaccumulation and biotransformation abilities between species and their hybrids should be evaluated to assess the comparability of the results obtained within the international biomonitoring programs. The objective of this study was to characterize bioaccumulation abilities and biomarker responses in Mytilus edulis, Mytilus galloprovincialis and their hybrids via an in situ transplantation experimentation on their progenies. Four mussel groups (M. edulis, M. galloprovincialis and two hybrids batches) issued from genetically characterized parents were transplanted for one year in Charente Maritime (France) to ensure their exposure to identical sources of contamination. The bioaccumulation of several families of contaminants (trace metals, polycyclic aromatic hydrocarbons, polybrominated diphenyl ethers, polychlorinated biphenyls), the response of several biomarkers (DNA strand breaks level, lysosomal membrane stability, metallothionein content, acetylcholine esterase activity) and some physiological parameters (growth, mortality, gonadal development), were analyzed. Differences were observed between species, however they were contaminant-specific. Variations in contaminants levels were observed between progenies, with higher levels of Cu, PBDE, PCB in M. edulis, and higher levels of Cd, Hg, Zn in M galloprovincialis. This study demonstrated that variations in contaminant bioaccumulation and different biomarker responses exist between Mytilus species in the field. Data on species or the presence of hybrid individuals (or introgression) is an important additional parameter to add to biomonitoring programs databases.

Replicated anthropogenic hybridisations reveal parallel patterns of admixture in marine mussels.

Human-mediated transport creates secondary contacts between genetically differentiated lineages, bringing new opportunities for gene exchange. When similar introductions occur in different places, they provide informally replicated experiments for studying hybridisation. We here examined 4,279 Mytilus mussels, sampled in Europe and genotyped with 77 ancestry-informative markers. We identified a type of introduced mussels, called "dock mussels," associated with port habitats and displaying a particular genetic signal of admixture between M. edulis and the Mediterranean lineage of M. galloprovincialis. These mussels exhibit similarities in their ancestry compositions, regardless of the local native genetic backgrounds and the distance separating colonised ports. We observed fine-scale genetic shifts at the port entrance, at scales below natural dispersal distance. Such sharp clines do not fit with migration-selection tension zone models, and instead suggest habitat choice and early-stage adaptation to the port environment, possibly coupled with connectivity barriers. Variations in the spread and admixture patterns of dock mussels seem to be influenced by the local native genetic backgrounds encountered. We next examined departures from the average admixture rate at different loci, and compared human-mediated admixture events, to naturally admixed populations and experimental crosses. When the same M. galloprovincialis background was involved, positive correlations in the departures of loci across locations were found; but when different backgrounds were involved, no or negative correlations were observed. While some observed positive correlations might be best explained by a shared history and saltatory colonisation, others are likely produced by parallel selective events. Altogether, genome-wide effect of admixture seems repeatable and more dependent on genetic background than environmental context. Our results pave the way towards further genomic analyses of admixture, and monitoring of the spread of dock mussels both at large and at fine spacial scales.

Genotoxicity in the rivers from the Brantas catchment (East Java, Indonesia): occurrence in sediments and effects in Oreochromis niloticus (Linnæus 1758).

This paper reports the first data from an integrated study investigating genotoxicity in the Brantas River, Java, Indonesia. Results showed that organic sediment extracts from the sites in the Brantas Delta retained genotoxic compounds identified using the SOS Chromotest and that the Aloo River and, to a lesser extent, the Surabaya River were the most contaminated studied sites. This genotoxicity was attributable to compounds that did not require any bioactivation under the test conditions. Occurrence of genotoxic effects was further investigated in erythrocytes from Nile tilapia, Oreochromis niloticus. High numbers of micronuclei were counted, especially in fish sampled in the rivers of the Brantas Delta. Moreover, cytoplasmic alterations which could be indicative of the presence of lipofuscin were found in the cytoplasm of the fish blood cells, especially in fish from the Aloo, Surabaya and Kalimas rivers. Altogether, our data showed that genotoxicity is occurring in fish living in rivers of the delta of the Brantas River and suggest that sediments from these sites may constitute a major source of pollution and hazard for species living or feeding in the area.

An Integrated Biomarker Approach Using Flounder to Improve Chemical Risk Assessments in the Heavily Polluted Seine Estuary.

The objective of this study was to develop an integrative approach in ecotoxicology (from biomarkers to population genetics) to assess the ecological status of fish populations. Flounders (Platichthys flesus) collected after the spawning season in the heavily polluted Seine estuary were compared with the moderately polluted Bay of Douarnenez. The muscle energetic reserves were highly depleted in Seine vs. Douarnenez fish. The Seine fish displaying a reduced capacity to manage the oxidative stress and a higher energetic metabolism. An increase in the content of muscle membrane phospholipids (sphingomyelin, phosphatidylserine, free sterols) was detected in the Seine vs. Douarnenez fish. The data integration allowed to hypothesize relationships between membrane phospholipids, xenobiotic metabolism, bioenergetics, and antioxidant defence. The genetic diversity considering neutral markers was maintained in the heavily polluted Seine population compared with the Douarnenez population. Finally, we suggest that the high physiological cost of tolerance to toxicants in the Seine flounder population could compromise its capacity to respond in the future to an additional stressor like warming waters in shallow depth. Thus, this population could be submitted to an ecological risk.

Coupling caging and proteomics on the European flounder (Platichthys flesus) to assess the estuarine water quality at micro scale.

Estuaries are important areas highly vulnerable to anthropogenic pollutions. Therefore, the assessment of estuarine water quality is a major ecological issue. In this study, we sampled juveniles of the European flounder in the "pristine" Canche estuary, and caged them in Canche and in two polluted sites of the Seine estuary, Rouen and Fosse Nord. After one month, the metal and organic pollutants in these sites were assessed, and we evaluated several phenotypic indicators (condition index, RNA/DNA ratios and genotoxicity), and extracted the proteins in fish livers for analysis using a shotgun proteomics approach. The results showed strong modifications in the fish caged in both sites of the Seine estuary, as compared to those caged in Canche. In particular, many proteins involved in phase I and phase II detoxification reactions were accumulated in the liver of fish caged in the site showing the highest pollution, Rouen. In addition, we observed a general disruption of metabolism, in particular an increase in lipid synthesis and carbohydrate degradation in Rouen, and a decrease in the abundance of proteins associated to translational activity in Fosse Nord. At both sites, several stress proteins were decreased. The proteomic impact of the encagement by itself was also evaluated, by comparing the liver proteome of fish caged in Canche to that of fish stayed in natura during the same time. The results showed proteomic signatures of exposure to stressful conditions (particularly heat stress), most probably related to the micro-habitat in which the cages were placed. In conclusion, the caging technique is of great interest for ecotoxicological assessment of estuarine waters, but should consider that the results are representative of the micro-habitat around the cages, which does not necessarily represent the overall heterogeneity of the estuarine environment.

Predictive Value of NRAMP1 and HGPX1 Gene Polymorphism for Maintenance BCG Response in Non-muscle-invasive Bladder Cancer.

AIM: To assess the potential predictive value of natural resistance-associated macrophage protein 1 (NRAMP1) and human glutathione peroxidase 1 (hGPX1) polymorphism in non-muscle-invasive bladder cancer treated with bacillus Calmette-Guerin (BCG) instillation, we conducted an original ancillary multicenter study. PATIENTS AND METHODS: We evaluated patients included in the multicenter URO-BCG 4 trial, who received three weekly instillations of one-third dose BCG every 6 months (group I) or two weekly instillations every 3 months (group II) for 3 years. For clinical evaluation we also evaluated tumor recurrence and muscle progression. NRAMP1 and hGPX1 polymorphism analyses were performed on blood DNA. NRAMP1 exon 15 and hGPX1 exon 1c were amplified using Type-it Microsatellite PCR Kit® for multiplex polymerase chain reaction. RESULTS: From June 2004 to April 2010, 146 randomized patients were included in this retrospective study. Blood samples were obtained from 107 patients. With 36 months of follow-up, 13.6% of patients had a tumor recurrence and muscle-invasive progression was observed in 4.3% of patients. Concerning NRAMP1 D543N polymorphism, patients with allele A had no tumor recurrence or muscle-invasive progression. No significant difference was observed in gene polymorphism distribution between groups I and II. Moreover, we did not observe any significant association of gene polymorphisms, tumor recurrence or muscle-invasive progression, event time and disease-free survival. CONCLUSION: Our results suggest that no significant difference was found for NRAMP1 and hGPX1 gene polymorphisms associated with recurrence time, muscle invasion frequency and disease-free survival, nevertheless, we observed that the NRAMP1 D543N GG genotype group had a shorter time to tumor recurrence.

TP53 and FGFR3 Gene Mutation Assessment in Urine: Pilot Study for Bladder Cancer Diagnosis.

AIM: To assess, in a prospective clinical research study, a new non-invasive and reliable test to accurately detect tumor protein 53 (TP53) and fibroblast growth factor receptor-3 (FGFR3) mutations in cells in urine. MATERIALS AND METHODS: TP53 mutations were analyzed using the functional analysis of separated allele in yeast (FASAY) method, which allows functional analysis of the P53 protein, and FGFR3 mutations were assessed with the SNaPshot system, detecting the eight most frequent point-mutations of this gene. Chi-square test or Fisher's exact test were used to compare TP53 and FGFR3 mutations in the tumors according to tumor stage and grade. RESULTS: TP53 and FGFR3 mutations in bladder tumors increased and decreased respectively with increasing tumor stage and cellular grade (p<0.05 and p<0.001, respectively). A total of 103 tumor/urinary sediment couples were analyzed. TP53 or FGFR3 mutations were observed in 76 tumors. The sensitivity for the detection of this type of mutation in urine was 46%, the specificity was 81%, the positive predictive value was 94% and the negative predictive value was 37%. CONCLUSION: Our original data confirmed the feasibility of TP53 and FGFR3 mutation detection in urine sediment. These measurements, together with urine cytology, may increase tumor detection. The sensitivity of the TP53/FGFR3 phenotype test in the urine was less than 50% and was not able to replace standard cystoscopy in the diagnosis of bladder tumors.

Cellular immune responses and phagocytic activity of fishes exposed to pollution of volcano mud.

Since May 29, 2006, a mud volcano in the Brantas Delta of the Sidoarjo district has emitted mud that has inundated nearby villages. Pollution in this area has been implicated in detrimental effects on fish health. In fishes, leukocyte and phagocytic cells play a vital role in body defenses. We report for the first time the effect of "LUSI" volcano mud on the immune systems of fish in the Brantas Delta. The aim of this study was to find biomarkers to allow the evaluation of the effects of volcanic mud and anthropogenic pollution on fish health in the Brantas Delta. The study took places at the Brantas Delta, which was polluted by volcano mud, and at reference sites in Karangkates and Pasuruan. Leukocyte numbers were determined using a Neubauer hemocytometer and a light microscope. Differential leukocyte counts were determined using blood smears stained with May Grunwald-Giemsa, providing neutrophil, lymphocyte and monocyte counts. Macrophages were taken from fish kidney, and their phagocytic activity was measured. In vitro analyses revealed that leukocyte and differential leukocyte counts (DLC) were higher in Channa striata and Chanos chanos caught from the polluted area. Macrophage numbers were higher in Oreochromis mossambicus than in the other species, indicating that this species is more sensitive to pollution. In areas close to volcanic mud eruption, all specimens had lower phagocytic activity. Our results show that immune cells were changed and phagocytic activity was reduced in the polluted area indicating cytotoxicity and alteration of the innate immune system in fishes exposed to LUSI volcano mud and anthropogenic pollution.

In vitro study of the effects of cadmium on the activation of the estrogen response element using the YES screen.

Estrogenic potential of environmental samples is frequently assessed using receptor-based functional assays. Using the yeast estrogen screen (YES) developed by Routledge and Sumpter, we assessed the ability of cadmium to activate the estrogen receptor-mediated response. No induced transcriptional activity was observed with a range of CdCl2 concentrations (1 nm-1mM). But, when combining cadmium with the model compound 17beta-estradiol, cadmium was able to significantly potentiate the induced estrogenic response for concentrations ranging from 15 nM to 1 microM. A maximal effect was observed at 0.5 microM with a ten fold reduction of the 17beta-estradiol EC50.