Towards a better consideration of endocrine disruption within the technical guidance for deriving environmental quality standards.

Endocrine-disrupting chemicals (EDCs) are a reason for growing concern because of their substantial and long-lasting deleterious effects on human health and wildlife populations. These include direct effects on aquatic organisms and may be a concern to species feeding on the aquatic food chains and water, including humans. In the European Community, the dedicated legislative tools to protect the aquatic environment and human health from contaminants released to surface waters is the Water Framework Directive (WFD). The achievement of protection goals is assessed through the comparison of concentrations measured in the media and thresholds of no effect called Environmental Quality Standards (EQSs). As EDCs are explicitly mentioned in the WFD, an analysis of the state of the art was undertaken on how far and how consistently ED properties were considered in the derivation of EQS values. Our results reveal substantial heterogeneity according to substance and that among substances with ED evidences, EQSs have been derived without considering ED properties for 70% of them. A methodology to better consider endocrine disrupting properties is proposed and includes a logical and systematic approach to derive EQSs with a proposal to specify additional assessment factors based on the specific hazard and potential uncertainty.

Effects of 17α-ethinylestradiol on the neuroendocrine gonadotropic system and behavior of European sea bass larvae (Dicentrarchus labrax).

The widespread use of 17α-ethinylestradiol (EE2), and other estrogenic endocrine disruptors, results in a continuous release of estrogenic compounds into aquatic environments. Xenoestrogens may interfere with the neuroendocrine system of aquatic organisms and may produce various adverse effects. The aim of the present study was to expose European sea bass larvae (Dicentrarchus labrax) to EE2 (0.5 and 50 nM) for 8 d and determine the expression levels of brain aromatase (cyp19a1b), gonadotropin-releasing hormones (gnrh1, gnrh2, gnrh3), kisspeptins (kiss1, kiss2) and estrogen receptors (esr1, esr2a, esr2b, gpera, gperb). Growth and behavior of larvae as evidenced by locomotor activity and anxiety-like behaviors were measured 8 d after EE2 treatment and a depuration period of 20 d. Exposure to 0.5 nM EE2 induced a significant increase in cyp19a1b expression levels, while upregulation of gnrh2, kiss1, and cyp19a1b expression was noted after 8 d at 50 nM EE2. Standard length at the end of the exposure phase was significantly lower in larvae exposed to 50 nM EE2 than in control; however, this effect was no longer observed after the depuration phase. The upregulation of gnrh2, kiss1, and cyp19a1b expression levels was found in conjunction with elevation in locomotor activity and anxiety-like behaviors in larvae. Behavioral alterations were still detected at the end of the depuration phase. Evidence indicates that the long-lasting effects of EE2 on behavior might impact normal development and subsequent fitness of exposed fish.

In vitro and in vivo studies of the endocrine disrupting potency of cadmium in roach (Rutilus rutilus) liver.

Cadmium has been reported to exert estrogenic, antiestrogenic or both effects in vertebrate species. To elucidate the endocrine disrupting action of CdCl2, ex vivo and in vivo experiments were performed in roach (Rutilus rutilus). Roach liver explants were exposed to a range of CdCl2 concentrations alone (0.1-50µM) or with an effective concentration (100nM) of 17ß-estradiol (E2). In addition, juvenile roach were intraperitoneally injected with CdCl2 (0.1-2.5mg/kg) with or without 1mg E2/kg. Subsequent analysis evaluated the effect of CdCl2 on vitellogenin (VTG) synthesis both at the mRNA and protein level, on estrogen receptors (erα and erß1) and on androgen receptor (ar) mRNA expression. Ex vivo and in vivo experiments indicated that CdCl2 is strongly anti-estrogenic as, when co-exposed to E2, CdCl2 significantly inhibited VTG production as well as vtg and erα mRNA expressions. Moreover, CdCl2 compromised the E2-mediated induction of the ar mRNA expression in vivo.

Intersex in the clam Scrobicularia plana (Da Costa): Widespread occurrence in English Channel estuaries and surrounding areas.

Estuarine clams Scrobicularia plana were sampled from 108 intertidal locations around the English Channel and adjacent areas. Although S. plana is believed to be a strict gonochorist, 58% of the populations sampled included intersexed individuals (described as male clams exhibiting ovotestis). Over the entire region, on average, 8.6% of male clams exhibited intersex, although proportions of affected males ranged from 0% to 53% depending on location. The severity of intersex was assessed using a simple classification scale, with the majority of individuals showing low levels of impact. Sex ratios were significantly skewed at some sites. There were no significant relationships between incidence or severity of intersex; or with size or parasitism of individual clams. Intersex in S. plana is a useful tool to assess endocrine disruptive effects in estuaries, although mechanisms of impact and causative agents remain uncertain.

Evidence of altered fertility in female roach (Rutilus rutilus) from the River Seine (France).

A large variety of anthropogenic chemicals present in the aquatic environment have been shown to be able to alter the endocrine system of exposed organisms, potentially impacting their reproductive function. The aim of this study was to assess the effects of environmental pollution on the reproductive system of wild female roach (Rutilus rutilus) from the Seine River (Normandy, France). A suite of biomarkers of endocrine disruption including gonado-somatic index, plasmatic vitellogenin, gonadal aromatase activity and histological parameters (oocyte diameter and gonad maturation) were studied. Female fish from the polluted sites showed a number of reproductive alterations, including inhibited gonad maturation, reduced oocyte growth, reduced levels of plasmatic vitellogenin and 3-fold lower gonadal aromatase activity than females collected in the reference site. Overall, these results highlight the presence of endocrine disruption in female roach from the Seine River.

In vivo effects of environmental concentrations of produced water on the reproductive function of polar cod (Boreogadus saida).

Offshore oil and gas drilling processes generate operational discharges such as produced water (PW), a complex mixture of seawater with polycyclic aromatic hydrocarbons (PAH) and alkylphenols (AP). Some of these compounds may interact with the endocrine system of marine organisms and alter reproductive functions. In this study, polar cod were exposed for up to 28 d to a mixture of PAH, alkylated PAH, and AP simulating the composition of North Sea PW, at low and high concentrations (1:2000 and 1:1000 dilution of the original concentrate, respectively). Potential adverse effects of PW on polar cod physiology were investigated through biomarkers of biotransformation (hepatic ethoxyresorufin O-deethylase [EROD] activity and bile PAH metabolites), endocrine disruption (plasma vitellogenin [VTG] levels and sex steroid concentrations), and gonad histology. Plasma sexual steroid levels in fish were not markedly affected by PW exposure, while higher plasma VTG concentrations were measured in females exposed to the high PW treatment for 7 and 28 d. In males exposed to the higher PW concentration, inhibition of spermatogenesis was observed after 28 d in addition to increase of melano-macrophage occurrence in testis. Females exposed to the high PW treatment for 21 d showed a significant increase of atresia incidence. Finally, a significant decrease in oocyte number was observed in high PW exposed female ovaries after 28 d of exposure.

[Preliminary study of p53 and FGFR3 gene mutations in the urine for bladder tumors]. / Étude préliminaire des mutations des gènes p53 et FGFR3 sur le culot urinaire des tumeurs de la vessie.

INTRODUCTION: Two major pathways are described in bladder carcinogenesis: one for invasive or high grade tumors characterized by alteration of the p53 tumor suppressor gene and the other for non-invasive tumors or low grade involving mutations FGFR3. The objective of our study was to validate the research in the urine of mutations in these two genes in patients with a bladder tumor. PATIENTS AND METHODS: In our preliminary study, we investigated 36 patients the FGFR3 and p53 mutations in tumors and urine collected during endoscopic resection. The p53 mutations were sought in FASAY, which allows a functional analysis of the protein P53. The FGFR3 mutations were sought in SNaPshot that searches the eight most frequent mutation points of this gene. RESULTS: For 24 patients (66% of cases), we found at least one of the two mutations in the tumor. This mutation was present in the urine in 15 patients (sensitivity=62.5%). In only one patient, we found a mutation in the urinary sediment that did not exist in the tumor (specificity=91.7%). CONCLUSION: The search for mutations of p53 and FGFR3 in the urine was a simple and non-invasive assay, which seems superior to urinary cytology for the detection of bladder tumors, raising hopes of an interest in this bio-assay for surveillance of bladder tumors and screening risk populations.

Physiological status and intersex in the endobenthic bivalve Scrobicularia plana from thirteen estuaries in northwest France.

The bivalve Scrobicularia plana, an important species for the structure and functioning of estuarine and coastal mudflats, was studied in thirteen sites from NW France differing by their degree of contamination to document the presence of reproduction impairments (intersex, sex ratio, gonadosomatic indices) in relation to the condition revealed by using hepatosomatic and condition indices. In agreement with recent studies in other European estuaries, intersex was revealed in all the studied estuaries, including sites the chemical and ecological status of which is considered "good" according to the criteria of the European Water Framework Directive. The presence of endocrine disrupting chemicals (EDCs) could result in such disturbances. Our results re-inforce the concern linked to the subtle effects of EDCs, which are active at very low doses, often in the absence of any major sign of toxicity. However at this stage, no clear link may be established between intersex and population effects.

A multibiomarker approach in juvenile turbot, Scophthalmus maximus, exposed to contaminated sediments.

Juvenile turbot were exposed in laboratory conditions to a mixture of chemical contaminants associated with harbour and estuarine sediments for seven and 21 days. Several molecular biomarkers of exposure were then measured in fish liver: two biotransformation parameters [ethoxyresorufin-O-deethylase (EROD) and phase II glutathione S-transferase (GST) activities] and an antioxidant enzyme activity [catalase (CAT)]. Modifications at the histological level were analysed by the measurement of the number and size of melanomacrophage centres (MMCs) and disturbances to the immune function by the measurement of cytokine transforming growth factor-beta (TGF-ß) and development of the thymus. The responses of these molecular and immunological biomarkers were correlated with metal and PAH concentrations measured in sediments and with the physiological performance of turbot analysed in a previous study on the same fish (growth rates, condition factor K, RNA:DNA ratio and lipid index). While no difference was found in thymus analysis, some molecular and immunological responses were observed in fish exposed to contaminated sediments. Weak relationships between molecular biomarkers' responses and PAH concentrations were recorded, while their responses were significantly correlated with some metals. MMC and aggregates were weakly related to chemical contaminants whereas some significant correlations were found between TGF-b1 responses and some metal concentrations. However, molecular and immunological biomarkers were weakly related to fish physiological damages since low responses were observed in the condition which led to the lowest growth and condition indices. These data suggest the complexity of cause-effect relationships between exposure to pollutants, metabolisms and health damages. Precautions should be considered in the use of molecular and immunological biomarkers alone in biomonitoring programs. Their complementary use with physiological biomarkers, such as fish growth and condition indices, could improve their utilisation.

Ligula intestinalis infection is associated with alterations of both brain and gonad aromatase expression in roach (Rutilus rutilus).

The tapeworm Ligula intestinalis commonly infests roach (Rutilus rutilus) and is responsible for the inhibition of gonad development. In order to better understand the effect of the plerocercoid on fish physiology, and to discriminate parasitization effects from those of endocrine-disrupting compounds (EDC), Cyp19b and Cyp19a aromatase expression was investigated by real-time quantitative polymerase chain reaction (PCR) in brain and gonads of ligulosed roach, caught from a reference site. Data were compared to reproductive and endocrine endpoints previously reported in a larger cohort study (including the sampled population of the present one), such as gonadosomatic index, Fulton index, gonadal histology, plasma sex steroid levels and brain aromatase activity. A decrease in Cyp19b expression in the brain of infected fish was demonstrated, in agreement with the reduction of aromatase activity previously described. In contrast, Cyp19a expression in the gonads appeared to be enhanced in ligulosed fish, in accordance with the presence of immature but differentiated sexual tissues. Together these results show that: (1) L. intestinalis infestation results in an alteration of aromatase expression which, in particular, may have profound effects on the fish brain; and (2) L. intestinalis infection must be considered as a major confounding factor in ecotoxicological studies using aromatase expression as an EDC biomarker. Moreover, the concordance between activity and expression--investigated for the first time in the same population--gives a functional relevance to the transcript aromatase dosage in the brain. Finally, quantitative PCR was confirmed as a sensitive approach, enabling aromatase status to be defined in the poorly developed gonads of ligulosed individuals.

Endocrine effects of the tapeworm Ligula intestinalis in its teleost host, the roach ( Rutilus rutilus).

The effects of parasite infection by the cestode Ligula intestinalis on the reproductive function and endocrine system of wild roach Rutilus rutilus were evaluated. Gonad maturation, plasma vitellogenin, plasma steroid concentrations (i.e. progesterone, 11-keto-testosterone and 17-beta-estradiol) and brain aromatase activity were investigated in relation with parasitization. A low prevalence (8%) of ligulosed roach and a moderate impact of parasitization (mean parasitization index of 8.8%) were found in the studied population. Inhibition of gonad maturation generally resulted from infestation but 5% of the ligulosed roach nevertheless reached maturity. Main sex steroid plasma content was depleted in both genders. Male 11-keto-testosterone, female 17-beta-estradiol and progesterone plasma concentrations of both genders were, respectively, 27, 5 and 3 times lower in ligulosed fish when compared to their non-infected counterparts. Progesterone levels were negatively correlated with the parasitization index in females. Brain aromatase activity of infected roach was reduced to 50% of that of the non-infected fish. These results demonstrate significant negative effects on the reproductive function of wild roach infected by the tapeworm L. intestinalis collected from a site with low contamination.

Biological indices, energy reserves, steroid hormones and sexual maturity in the infaunal bivalve Scrobicularia plana from three sites differing by their level of contamination.

The aim of this work was to investigate in situ biometric (condition index, hepato-somatic index, gonado-somatic index), biochemical (glycogen, lipids, sexual steroids) and histological (sex, sexual maturation stage) variables in the infaunal bivalve Scrobicularia plana. First, the reproductive cycle of S. plana was assessed by collecting bivalves from a reference site (the bay of Bourgneuf, Fr) in April, May, July, September and November 2005 and in January and March 2006. Then, S. plana were collected at three key periods of their sexual cycle (March 2006, beginning of gametogenesis; June 2006, spawning; and September 2006, spent) in three sites differing by their level of contamination (bay of Bourgneuf, reference site; Loire and Seine estuaries; Fr, impacted sites). The reproductive cycle of S. plana was well defined with a clear spawning period between May and July, sexual repose from November to January. Development of the gonad began in January and ended in September. Sex-ratio was determined during spawning and the influence of sex on biochemical variables was examined. Progesterone, 17beta-estradiol and testosterone levels in the gonad of S. plana were close to those reported in other bivalves. This study is the first to demonstrate in situ influence of site, sex and sexual maturity on energy reserves, and sexual steroids in S. plana. Even if interpretation of results is complex due to interferences between natural and anthropogenic factors, S. plana is a suitable species for estuarine studies and a better understanding of its reproduction will permit to assess impacts of environmental pollutants.

Environmental risk assessment: a critical approach of the European TGD in an in situ application.

The aim of this study was to test field relevance of the prospective methodology for the assessment of environmental risk described in the EU technical guidance document (TGD) [European Commission 2003. Technical guidance document in support of Commission Directive 93/67/EEC on Risk assessment for new notified substances and Commission Regulation (EC) No 1488/94 on Risk assessment for existing substances and Commission Directive (EC) 98/8 on biocides, second ed. European Commission, Luxembourg, Part 1, 2 and 3, 760 pp.]. To achieve this goal, an environmental risk assessment was performed according to the TGD for two major contaminants, atrazine and diuron, that are present in the Seine River estuary (France) and listed in the EU Water Framework Directive (Directive 2000/60/CE). Results showed that atrazine presented a source of risk in the upper- and mid-estuary throughout the 1993 and 1996 spring seasons. Diuron introduced a risk into the same areas throughout spring periods of 1993-2005. Results are discussed and some suggestions for a more realistic in situ risk assessment are given. For the computation of a more relevant PNEC for pesticides, their specific mode of action should be taken into consideration as well as ecotoxicological data on species endemic to the considered area.

Detection of environmental mutagens using the FACIM assay.

A genetically engineered diploid yeast strain named yJC2, was specifically developed for environmental mutagen detection and characterization of induced mutations. This strain contains one copy of the human TP53 tumour suppressor gene coding sequence which is used as a molecular target for mutagens and two copies of the ADE2 reporter gene allowing accurate measurement of the TP53 transcriptional activity. The strain sensitivity to mutagens was evaluated by exposing cells to UVC, 4-nitroquinoline (NQO) or to an organic extract of sediment from the Seine estuary. For all studied mutagens, a significant and dose-dependent increase of mutant frequency was observed. The present assay named FACIM II (Functional Analysis of Chemical-Induced TP53 Mutations) is more convenient than the FACIM I and more inducible than the SOS Chromotest to detect direct-acting mutagens in the environment.

Profiles of environmental and endogenous estrogens in the zebra mussel Dreissena polymorpha.

Contamination of freshwater environments by estrogenic compounds has led to concern over potential impacts on invertebrate species. The uptake of the environmental estrogen 17beta-estradiol (E2) by the freshwater bivalve Dreissena polymorpha and the nature of estrogenic substances in tissues of D. polymorpha mussels collected from four freshwater sites were investigated. Exposure of mussels to [(14)C]-E2 (7.5 ngl(-1), 13 days) revealed that the estrogen bioconcentrated 840+/-58 (males) and 580+/-77 (females) fold (mean+/-95% confidence limits) and was metabolised in tissues to a persistent lipophilic ester. Estrogenic activity, measured using a recombinant human estrogen receptor transcription screen (YES), was detected in tissue extracts of all mussels sampled from freshwater sites. At two reference sites the estrogenic activities of mussel tissues were <1ng E2 equivalents g(-1) wet weight tissue (ng EEQ g(-1) ww) which increased to 7.4-45.7ng EEQg(-1) ww for both free and esterified estrogens extracted from hydrolysed tissue extracts. In mussels collected from two contaminated river sites, estrogenic activity was 0.2-6.7ng EEQ g(-1) ww (free estrogens) and 25.6-316.2ng EEQ g(-1) ww for total estrogens. Fractionation of the tissue extracts revealed that E2 (as the ester) was the predominant estrogen detected in both sexes of D. polymorpha, however, the xenoestrogen nonylphenol (NP) was also detected in mussels sampled from contaminated rivers. The detection of endogenous esterified E2 and the potential for accumulation of exogenous E2 and NP in D. polymorpha tissues suggests that this bivalve could be susceptible to exposure to estrogenic contaminants in the aquatic environment.

Seasonal variations of a battery of biomarkers and physiological indices for the mussel Mytilus galloprovincialis transplanted into the northwest Mediterranean Sea.

Seasonal variations of six mussel (Mytilus galloprovincialis) biomarkers at two sites in the Mediterranean Sea were compared with physiological indices (condition, growth and gonad maturation), environmental parameters (temperature, salinity and turbidity), and chemical contamination levels. The basal levels of acetylcholinesterase (AChE), DNA adducts, benzo[a]pyrene hydroxylase (BPH), heat-shock proteins (HSP70), metallothioneins (MT) and P-glycoprotein (P-gp)-mediated multixenobiotic resistance (MXR) were estimated as early warning signals in caged mussels sampled at Carteau (native site) and La Fourcade (transplantation site) over a 2-year period. The Carteau and La Fourcade mussels have specific chemical contamination profiles but a similar range of values. For example, both are highly contaminated by heavy metals (201 and 258.4 mg kg(-1) dw, respectively) and considered as moderately impacted for polychlorinated biphenyls (PCBs) and polycyclic aromatic hydrocarbons (PAHs). However, contamination levels at Carteau are twice as high for PAHs (101.5 mg g(-1) dw) and PCBs (90.2 mg g(-1) dw) than La Fourcade. The seasonal contamination trend at Carteau showed six-fold higher levels of pyrolytic pollutants in winter. Although few tissue lesions were detected in individuals studied at either site, greater parasitic infestation was observed at Carteau. The results of findings from the two Mediterranean pilot studies support the adaptability of transplanted mussels to be used as biomarkers and to establish physiological endpoints for chemical contaminant exposure.

Biochemical and histological responses in mussel (Mytilus edulis) exposed to North Sea oil and to a mixture of North Sea oil and alkylphenols.

Several environmental chemicals are suspected to be responsible for adverse health effects on the reproductive system in various organisms. During this work, environmentally relevant concentrations of North Sea oil were used alone or in combination with alkylphenols and additional PAH to study the effect on vitellogenin-like protein expression and gonadal development in mussels. North Sea oil (0.5 ppm) induced the expression of phospho-proteins in both sexes indicating that some compounds are oestrogen-mimics. This induction was not seen in samples dosed with the mixture but signs of toxic effects were observed in the gonads. Indeed, numerous degenerating ovarian follicles in females and foci, similar to vertebrate melanomacrophage centres, were observed in testes.

Expression and activity of a multixenobiotic resistance system in the Pacific oyster Crassostrea gigas.

The multidrug resistance (MDR) mechanism corresponds to a defence system relying on the expression of high molecular membrane proteins that can actively lower the intracellular concentration of a wide variety of toxins, thus maintaining them below their toxic level. Using RT-PCR, expression levels of a gene belonging to the class I of mammalian mdr genes, has been assessed in different developmental stages of the oyster Crassostrea gigas. While no expression was found in the oocyte or the trocophore stage, a rise of mRNA content was observed from the veliger stage to the juvenile stage, thus indicating the induction of the system as the animal is developing in the environment. The incubation of gill fragments in the dye rhodamine B and subsequent measurements of intracellular fluorescence using a microplate reader indicates that the system can effectively decrease the accumulation of the test compound in a competitive manner with known inhibitors or environmental contaminants as observed in vertebrate cells. The oyster MXR system is thus becoming active in adult oyster and could be of importance in environmentally contaminated areas.

Genetic and immunological characterisation of a multixenobiotic resistance system in the turbot (Scophthalmus maximus).

Pleiotropic resistance driven by transport proteins constitutes a very ubiquitous protection mechanism against natural or synthetic toxic compounds. The multidrug (MDR) or multixenobiotic (MXR) system has been identified in many different species, and may be used as a biomarker for pollution assessment. Here we report the existence of a gene encoding a MXR-related protein in a benthic fish species, the turbot Scophthalmus maximus, and its constitutive expression in several tissues. A 433bp cDNA fragment has been cloned by RT-PCR. The deduced amino-acid sequence shares close to 80% homology with class I or class II mammalian MDR proteins. This cDNA corresponds to a major mRNA of 5.6 kb and encodes a protein having an apparent molecular weight of 83 kDa. Constitutive expression levels assessed by semi-quantitative RT-PCR and Western blot, revealed that the kidney and the brain, and to a lesser extent, the heart, gills and intestine, are the organs which contain the highest amount of both MXR mRNAs or proteins. This tissue specific expression suggests a role for the identified mechanism in protection against endogenous or exogenous toxic compounds.