Biomarkers Of Endocrine Disruption Research Articles

Environmental exposure to per- and perfluoroalkyl substances in early pregnancy and newborn anogenital distance: A prospective cohort study.

Per- and polyfluoroalkyl substances (PFAS) are persistent, ubiquitous pollutants, and the current epidemiological evidence regarding the impact of in utero exposure to PFAS on anogenital distance (AGD) is limited and inconclusive. The primary aim of this study was to investigate the potential associations between maternal exposure to PFAS during pregnancy and AGD in newborns. A total of 2273 mother-child pairs were recruited for this study, and both PFAS levels and AGD were measured. Multiple linear regression models were utilized to explore the relationships between individual PFAS and AGD. Additionally, quantile-based g-computation (QGC) was employed to assess the joint effects of mixtures of PFAS on AGD. Our findings showed that maternal exposure to PFOS (β = 0.518, 95% CI: 0.093, 0.942), PFNA (β = 0.487, 95% CI: 0.037, 0.937), PFDA (β = 0.443, 95% CI: 0.048, 0.838), PFUA (β = 0.434, 95% CI: 0.031, 0.838), and PFBS (β = 0.444, 95% CI: 0.124, 0.763) during early pregnancy had a significant positive association with AGD in boys. Similarly, in girls, maternal exposure to PFOS (β = 0.423, 95% CI: 0.006, 0.841), PFNA (β = 0.641, 95% CI: 0.207, 1.074), PFDA (β = 0.670, 95% CI: 0.306, 1.033), PFUA (β = 0.895, 95% CI: 0.509, 1.281), and PFBS (β = 0.474, 95% CI: 0.178, 0.770) had a positive association with AGD, while PFOA (β = -1.254, 95% CI: -1.786, -0.723) had a negative association. QGC models further confirmed that PFAS mixtures were positively associated with AGD. Moreover, PFBS was the primary contributor to the joint effects of PFAS mixtures on AGD. In summary, our study has provided further corroboration for the possibility that PFAS exposure can have an impact on AGD in both boys and girls. The use of AGD as a promising biomarker for endocrine disruption highlights the significance of our findings, which may have valuable clinical implications for reproductive diseases.

Physicochemical and bioinformatic characterization of Oreochromis niloticus vitellogenin as an endocrine disruption biomarker.

Aquatic biota is increasingly being exposed to chemical pollutants due to human activities and the relationship between the level of environmental pollution and fish reproduction is a continuously ongoing issue. The vitellogenin (Vtg) protein synthesis can be induced in the liver of juvenile and male fish after stimulation of the estrogen receptor and therefore, Vtg has been used as a biomarker of xenoestrogen exposure in several fish species. The current study reported the first physicochemical characterization of Vtg from Oreochromis niloticus. Adult male fish were exposed to 17α-ethinylestradiol for Vtg induction. Purified vitellogenin from plasma showed low stability at 25 and 4 °C in saline conditions, and good stability in acidic (low pH) or in heated conditions. The 3D modeling provided useful information on the structure of O. niloticus Vtg showing conserved structural features. According to bioinformatics and experimental results, there are important structural differences between the two chemical forms of Vtg (VtgAb and VtgC) in a phylogenetic context. The present results add information about the development of ecotoxicological immunoassays to study the endocrine disruption in O. niloticus improving the Vtg performance as a biomarker of reproduction in fish.

Transcriptome signatures of wastewater effluent exposure in larval zebrafish vary with seasonal mixture composition in an effluent-dominated stream

Wastewater treatment plant (WWTP) effluent-dominated streams provide critical habitat for aquatic and terrestrial organisms but also continually expose them to complex mixtures of pharmaceuticals that can potentially impair growth, behavior, and reproduction. Currently, few biomarkers are available that relate to pharmaceutical-specific mechanisms of action. In the experiment reported in this paper, zebrafish (Danio rerio) embryos at two developmental stages were exposed to water samples from three sampling sites (0.1 km upstream of the outfall, at the effluent outfall, and 0.1 km below the outfall) during base-flow conditions from two months (January and May) of a temperate-region effluent-dominated stream containing a complex mixture of pharmaceuticals and other contaminants of emerging concern. RNA-sequencing identified potential biological impacts and biomarkers of WWTP effluent exposure that extend past traditional markers of endocrine disruption. Transcriptomics revealed changes to a wide range of biological functions and pathways including cardiac, neurological, visual, metabolic, and signaling pathways. These transcriptomic changes varied by developmental stage and displayed sensitivity to variable chemical composition and concentration of effluent, thus indicating a need for stage-specific biomarkers. Some transcripts are known to be associated with genes related to pharmaceuticals that were present in the collected samples. Although traditional biomarkers of endocrine disruption were not enriched in either month, a high estrogenicity signal was detected upstream in May and implicates the presence of unidentified chemical inputs not captured by the targeted chemical analysis. This work reveals associations between bioeffects of exposure, stage of development, and the composition of chemical mixtures in effluent-dominated surface water. The work underscores the importance of measuring effects beyond the endocrine system when assessing the impact of bioactive chemicals in WWTP effluent and identifies a need for non-targeted chemical analysis when bioeffects are not explained by the targeted analysis.

Comprehensive assessment of chemical residues in surface and wastewater using passive sampling, chemical, biological, and fish behavioral assays

Effluents from ten full-scale municipal wastewater treatment plants (WWTPs) that discharge into the Hudson River, surface waters, and wild-caught fish samples were analyzed using liquid chromatography with tandem mass spectrometry (LC/MS/MS) to examine the influence of wastewater discharge on the concentrations of contaminants of emerging concern (CECs) and their ecological impacts on fish. Analysis was based on targeted detection of 41 pharmaceuticals, and non-targeted analysis (suspect screening) of CECs. Biological effects of treated WWTP effluents were assessed using a larval zebrafish (Danio rerio) swimming behavior assay. Concentrations of residues in surface waters were determined in grab samples and polar organic chemical integrative samplers (POCIS). In addition, vitellogenin peptides, used as biomarkers of endocrine disruption, were quantified using LC/MS/MS in the wild-caught fish plasma samples. Overall, 94 chemical residues were identified, including 63 pharmaceuticals, 10 industrial chemicals, and 21 pesticides. Eight targeted pharmaceuticals were detected in 100% of effluent samples with median detections of: bupropion (194 ng/L), carbamazepine (91 ng/L), ciprofloxacin (190 ng/L), citalopram (172 ng/L), desvenlafaxine (667 ng/L), iopamidol (3790 ng/L), primidone (86 ng/L), and venlafaxine (231 ng/L). Over 30 chemical residues were detected in wild-caught fish tissues. Notably, zebrafish larvae exposed to chemical extracts of effluents from 9 of 10 WWTPs, in at least one season, were significantly hyperactive. Vitellogenin expression in male or immature fish occurred 2.8 times more frequently in fish collected from the Hudson River as compared to a reference site receiving no direct effluent input. Due to the low concentrations of pharmaceuticals detected in effluents, it is likely that chemicals other than pharmaceuticals measured are responsible for the behavioral changes observed. The combined use of POCIS and non-target analysis demonstrated significant increase in the chemical coverage for CEC detection, providing a better insight on the impacts of WWTP effluents and agricultural practices on surface water quality.

On the Use and Interpretation of Areola/Nipple Retention as a Biomarker for Anti-androgenic Effects in Rat Toxicity Studies.

Areola/nipple retention (NR) is an established biomarker for an anti-androgenic mode of action in rat toxicity studies. It is a mandatory measurement under several OECD test guidelines and is typically assessed in combination with anogenital distance (AGD). Both NR and AGD are considered retrospective biomarkers of insufficient androgen signaling during the masculinization programming window in male fetuses. However, there are still aspects concerning NR as a biomarker for endocrine disruption that remains to be clarified. For instance, can NR be regarded a permanent adverse effect? Is it a redundant measurement if AGD is assessed in the same study? Is NR equally sensitive and specific to anti-androgenic chemical substances as a shortening of male AGD? In this review we discuss these and other aspects concerning the use of NR as a biomarker in toxicity studies. We have collected available literature from rat toxicity studies that have reported on NR and synthesized the data in order to draw a clearer picture about the sensitivity and specificity of NR as an effect biomarker for an anti-androgenic mode of action, including comparisons to AGD measurements. We carefully conclude that NR and AGD in rats for the most part display similar sensitivity and specificity, but that there are clear exceptions which support the continued assessment of both endpoints in relevant reproductive toxicity studies. Available literature also support the view that NR in infant male rats signifies a high risk for permanent nipples in adulthood. Finally, the literature suggests that the mechanisms of action leading from a chemical stressor event to either NR or short AGD in male offspring are overlapping with respect to canonical androgen signaling, yet differ with respect to other mechanisms of action.

Gene expression analysis on growth, development and toxicity pathways of male Nile tilapia (Oreochromis niloticus), after acute and sub-chronic benzo (α) pyrene exposures

Benzo[α]pyrene (BaP), a lipophilic polycyclic aromatic hydrocarbon (PAH), is a contaminant widely distributed in aquatic systems. Its presence in freshwater organisms is of great concern; particularly in Nile tilapia (Oreochromis niloticus), due to its economic relevance. The aim of this study is to evaluate the effects of acute and sub-chronic BaP exposures on molecular growth/development responses, toxicity to DNA pathways and xenobiotic metabolism. Negative morphometric changes (the growth condition factor, hepatosomatic and gonadosomatic indices), the fluorescent aromatic compounds (FACs) in bile were also studied in order to understand the mechanisms of action of BaP. Genes involved in the growth hormone GH/insulin-like growth factor 1 (IGF-1) were measured, such as IGF1–2 with the growth hormone receptor gene expression GHR1–2, and the endocrine disruption biomarker vitellogenin (VTG). Acute exposure elicited changes in the GH/IGF axis, mainly in the GHR1 and in IGF1 mRNA levels without affecting the GHR2 expression. While sub-chronic exposure had less effect on both GHR and IGF genes. The most notable tissue-specific effects and morphometric endpoints were observed upon sub-chronic exposure, such as changes in key genes involved in detoxification, DNA damage, and altered reproductive morphological endpoints; showing that sub-chronic BaP doses have longer-lasting toxic effects. This study shows that sub-chronic BaP exposure may compromise the health of Nile tilapia and sheds light on the changes of the GH/IGF axis and the biotransformation of the xenobiotics due to the presence of this contaminant.

Evaluation of the effect of carbamazepine on the concentration of vitellogenin in Pseudoplatystoma magdaleniatum

Abstract Carbamazepine is a pharmaceutical used in patients with seizures and bipolar disorder, which has been found in wastewater and many water resources. This is due to the inadequate disposal of pharmaceutical waste and the lack of treatment of municipal wastewater, as is the case in Colombia. The two main hydrographic basins of Colombia are the Cauca and Magdalena rivers, which are inhabited by the endemic species Striped Catfish (Pseudoplatystoma magdaleniatum). This has become an endangered species for various reasons, including the high contamination level of these rivers. In 2019, mature adult P. magdaleniatum of both sexes were caught in the Cauca river in Colombia. This was in order to assess the concentration of vitellogenin, as a biomarker of endocrine disruption, resulting from exposure to different levels of concentration of the emerging contaminant carbamazepine for 4 months. These tests were carried out in a fish farm. A significant decrease in the vitellogenin concentration was verified in females at concentrations of 25 µg l-1 and 50 µg l-1, and in males at 50 µg l-1 of carbamazepine, with a significance level of p˂0.05. Carbamazepine could cause a negative feedback in gonadotropin secretion, acting as an estrogen mimicker that causes a decrease in the level of vitellogenin.

Impact of Bisphenol S as an Endocrine Disruptor in a Freshwater Fish, Oreochromis mossambicus

Endocrine Disrupting Chemicals (EDCs) have the potential to alter the hormonal pathways concerned with regulation of the normal homeostatic mechanisms. The adverse effects of EDCs can be observed in areas where pollution is high, particularly in aquatic ecosystems, where persistent environmental chemicals accumulate. In the present study, we investigated the endocrine disrupting effects of Bisphenol S (BPS), if any, in the levels of thyroxine, triiodothyronine, cortisol and sex steroidal [17I²estradiol (E2) and testosterone(T)] hormones in juvenile as well as adult Oreochromis mossambicus . Drastic changes in serum thyroxine and triiodothyronine levels showed that BPS treatment resulted in disruption of thyroid gland function. Alteration to significant levels in serum cortisol indicated acute stress and impairment of hypothalamic-pituitaryinterrenal axis. Significant changes (p<0.05) occurred in the steroidal hormone levels which are biomarkers of endocrine disruption as they affect hypothalamic-pituitary-gonadal axis in fish. Bisphenol S also revealed estrogenic potency by inducing significant alteration in the E2/T ratio. DNA fragmentation, if any, induced by BPS was also analyzed in juvenile fish. There was no significant DNA fragmentation observed in gel electrophoresis, but a significant elevation was seen in percent fragmented DNA in the diphenylamine method. Therefore, it could be concluded that BPS at different sublethal concentrations have a profound impact on endocrine physiology of fish.

Vitellogenin gene expression in marine mussels exposed to ethinylestradiol: No induction at the transcriptional level

Vitellogenin (Vtg), a large multidomain protein precursor of egg-yolk proteins, is used as an endocrine disruption biomarker in fish, and in the last decades, its use has been extended to invertebrates like mollusks. However, it remains unclear whether invertebrate endocrine system produces Vtg in response to estrogens, like it occurs in oviparous vertebrates. In a previous study, no evidence of induction of Vtg expression at protein level was found in gonads of the marine mussel Mytilus galloprovincialis after exposure to the estrogenic chemical 17α-ethinylestradiol (EE2). In the present follow-up study, it was investigated whether there is any effect of EE2 on Vtg abundance at transcriptional level in M. galloprovincialis gonads. To this aim, RT-qPCR analysis targeting three different domains of Vtg transcript was performed on gonads of mussels that were exposed either 4 or 24 days to 100 ng/L EE2. In addition, several reference genes were analysed and a selection of these for potential use in further RT-qPCR analyses on mussel male and female gonads is provided. Results showed higher expression in females than in males for the three analysed Vtg domains, and no evidence of Vtg mRNA induction due to EE2 either in females or males. The present results, together with those obtained from previous analysis at protein level, support that Vtg is not an adequate biomarker for xenoestrogenicity in marine mussels. Additionally, nucleotide sequences of Vtg transcripts of three closely-related species from Mytilus edulis complex (M. galloprovincialis, M. edulis and M. trossulus) are provided and compared with Vtg sequences from other mollusk species to assess the level of conservation and evolutionary relationships among species.

No evidence that vitellogenin protein expression is induced in marine mussels after exposure to an estrogenic chemical

A wide variety of endocrine disrupting chemicals reach the marine environment and can cause harmful effects in different marine organisms. Vitellogenin (Vtg), the egg-yolk precursor, is a commonly used endocrine disruption biomarker in fish and more recently in marine invertebrates under the assumption of high expected similarities in the endocrine system of vertebrates and invertebrates. However, this assumption has been recently questioned. The results from previous studies focused on bivalve molluscs showed that Vtg induction could be misleading because of the use of either non-robust or indirect techniques to measure Vtg. In this study, mussels (Mytilus galloprovincialis) were exposed to either 10 or 100 ng/L of the synthetic hormone 17α-ethinylestradiol (EE2) at different exposure times (4 and 24 days) and under different feeding regimes (representing different energy balances), and Vtg levels in both male and female mussel gonads were quantified by label free shotgun LC-MS/MS proteomic analysis. Vtg protein was not detected in male gonads. In female gonads, Vtg levels were not significantly affected by EE2 at any exposure time or EE2 concentration tested, whereas a significant correlation was found between the degree of maturation of the gonad and Vtg levels in females. Results obtained in the present study critically question the use of Vtg as a biomarker of endocrine disruption in marine mussels, and show that the degree of maturation of the gonad can be an important confounding factor in the attempts to evaluate estrogenic effects through Vtg measurement in mussel gonads.

Endocrine and reproductive endpoints of Notothenia rossii and N. coriiceps: A baseline study for ecotoxicological monitoring in Antarctic waters

Pollution threats Antarctica and scientists blame xenobiotics and anthropic activities. Yet little is known about their effect on Antarctic ichthyofauna. Accordingly, we investigated the endocrine system of male fish Notothenia rossii and N. coriiceps (Perciformes, Nototheniidae) collected during Austral summer. For N. rossii, hormone levels of estradiol, testosterone, and 11-ketotestosterone were higher in fish collected during March than those of January; whereas for N. coriiceps estradiol and androgens levels were higher and lower in March, respectively. Histological analysis of the testes showed an unrestricted lobular type with no pathological alterations. However, detection of vitellogenin-like in plasma and skin mucus were seen in 75% of N. coriiceps males and 7% of N. rossii males. This is the first report of mucus vitellogenin-like detection as a non-invasive biomarker of endocrine disruption in notothenioid males and settles a baseline for future studies of reproductive biology and endocrine disruption in Antarctic fishes.

Wax esters of uropygial gland secretion as biomarkers of endocrine disruption in birds exposed to treated sewage water

The chemical composition of the uropygial gland secretion of birds shows seasonal, sex and age-related variations following sex hormones fluctuations. We explore the use of the composition of the uropygial gland secretion as a non-invasive biomarker of endocrine disruption in 137 common moorhens (Gallinula chloropus) from Navaseca Pond, which receives the effluent of a wastewater treatment plant, and from the more pristine Tablas de Daimiel National Park in Spain. Wax ester and fatty acid compositions were measured by means gas chromatography – mass spectrometry (GC-MS) in the uropygial gland secretion of moorhens from both wetlands. Organochlorine compounds (p,p’-DDE and PCBs) were measured in blood and uropygial gland secretion of moorhens as indicators of anthropogenic pollutants, and this information was interpreted together with previous results of the accumulation of metals and metalloids in blood and feathers of these moorhens and a wide range of endocrine disrupting chemicals (EDCs) measured in water from both study sites. PCBs and p,p'-DDE were found in 32% of the blood and 51% of uropygial gland secretion samples, being at highest levels in Navaseca. Wax composition was dominated by monoesters of 35–38 carbons and displayed a clear seasonal variation, in which long-chain wax esters were more abundant in spring-summer than in autumn-winter. This seasonal change was less evident in birds from Navaseca, where the presence of shorter wax esters was associated with the higher concentration of PCBs in uropygial gland secretion. The observed effect may not be associated with this specific type of pollutants because moorhens in Navaseca are also exposed to a wide diversity endocrine disruptors as shown in a previous study. Uropygial gland secretion can be a useful non-invasive sample for integrating chemical monitoring of pollutants and their effects as endocrine disruptors in birds.

Assessing the impact of waste water effluents on native fish species from a semi-arid region, NE Spain

Mediterranean rivers are strongly affected by pollution and water scarcity. Over the summer period, urban and industrial effluents arrive into the rivers with little dilution. In order to assess the water quality, two native fish species, Barbus meridionalis and Squalius laietanus, were collected from six sites along the Ripoll River (Spain). PAH metabolites, alkylphenols (nonylphenol and octylphenol) and the musk galaxolide levels were determined in bile. 7-Ethoxyresorufin-O-deethylase (EROD) and 7-benzyloxy-4-trifluoromethyl-coumarin O-debenzyloxylase (BFCOD) were measured as metabolic biomarkers, and the activity of CYP19 aromatase was determined in the ovaries of B. meridionalis as a biomarker of endocrine disruption. The analysis of bile indicated that fish from the lower course of the river were highly exposed to different pollutants. Accordingly, a significant induction of EROD (9 to 10-fold) and BFCOD (3 to 5-fold) activities were detected in both fish species together with an increased aromatase activity in females of B. meridionalis from the most polluted sites. Considering that sewage treatment plant (STP) effluents are essential for maintaining environmental flows in small Mediterranean rivers, this study highlights the need to improve the efficiency of STPs to protect fish health.

Molecular isolation and characterization of the kisspeptin system, KISS and GPR54 genes in roach Rutilus rutilus.

The reproduction of vertebrates is regulated by endocrine and neuro-endocrine signaling molecules acting along the brain-pituitary-gonad (BPG) axis. The understanding of the neuroendocrine role played in reproductive function has been recently revolutionized since the KiSS1/GPR54 (KiSS1r) system was discovered in 2003 in human and mice. Kisspeptins, neuropeptides that are encoded by the KiSS genes, have been recognized as essential in the regulation of the gonadotropic axis. They have been shown to play key roles in puberty onset and reproduction by regulating the gonadotropin secretion in mammals while physiological roles in vertebrates are still poorly known. In order to provide new knowledge on basic reproductive physiology in fish as well as new tools to assess impacts of endocrine disrupting compounds (EDCs), the neurotransmitter system, i.e., gene/receptor, KISS/GPR54 might constitute an appropriate biomarker. This study provides new understandings on the neuroendocrine regulation of roach reproduction as well as new molecular tools to be used as biomarkers of endocrine disruption. This work completes the set of biomarkers already validated in this species.

Relation of contaminants to fish intersex in riverine sport fishes

Endocrine active compounds (EACs) are pollutants that have been recognized as an emerging and widespread threat to aquatic ecosystems globally. Intersex, the presence of female germ cells within a predominantly male gonad, is considered a biomarker of endocrine disruption caused by EACs. We measured a suite of EACs and assessed their associated impacts on fish intersex occurrence and severity in a large, regulated river system in North Carolina and South Carolina, USA. Our specific objective was to determine the relationship of contaminants in water, sediment, and fish tissue with the occurrence and severity of the intersex condition in wild, adult black bass (Micropterus), sunfish (Lepomis), and catfish (Ictaluridae) species at 11 sites located on the Yadkin-Pee Dee River. Polycyclic aromatic hydrocarbons (PAHs), ethinylestradiol (EE2), and heavy metals were the most prevalent contaminants that exceeded effect levels for the protection of aquatic organisms. Fish intersex condition was most frequently observed and most severe in black basses and was less frequently detected and less severe in sunfishes and catfishes. The occurrence of the intersex condition in fish showed site-related effects, rather than increasing longitudinal trends from upstream to downstream. Mean black bass and catfish tissue contaminant concentrations were higher than that of sunfish, likely because of the latter's lower trophic position in the food web. Principal component analysis identified waterborne PAHs as the most correlated environmental contaminant with intersex occurrence and severity in black bass and sunfish. As indicated by the intersex condition, EACs have adverse but often variable effects on the health of wild sport fishes in this river, likely due to fluctuations in EAC inputs and the dynamic nature of the riverine system. These findings enhance the understanding of the relationship between contaminants and fish health and provide information to guide ecologically comprehensive conservation and management decisions.

Identification, expression, and endocrine-disruption of three ecdysone-responsive genes in the sentinel species Gammarus fossarum

Taking advantage of a large transcriptomic dataset recently obtained in the sentinel crustacean amphipod Gammarus fossarum, we developed an approach based on sequence similarity and phylogenetic reconstruction to identify key players involved in the endocrine regulation of G. fossarum. Our work identified three genes of interest: the nuclear receptors RXR and E75, and the regulator broad-complex (BR). Their involvement in the regulation of molting and reproduction, along with their sensitivity to chemical contamination were experimentally assessed by studying gene expression during the female reproductive cycle, and after laboratory exposure to model endocrine disrupting compounds (EDCs): pyriproxyfen, tebufenozide and piperonyl butoxide. RXR expression suggested a role of this gene in ecdysis and post-molting processes. E75 presented two expression peaks that suggested a role in vitellogenesis, and molting. BR expression showed no variation during molting/reproductive cycle. After exposure to the three EDCs, a strong inhibition of the inter-molt E75 peak was observed with tebufenozide, and an induction of RXR after exposure to pyriproxyfen and piperonyl butoxide. These results confirm the implication of RXR and E75 in hormonal regulation of female reproductive cycles in G. fossarum and their sensitivity towards EDCs opens the possibility of using them as specific endocrine disruption biomarkers.

Controversial use of vitellogenin as a biomarker of endocrine disruption in crustaceans: New adverse pieces of evidence in the copepod Eurytemora affinis

In recent years, the interest in the use of vitellogenin (VTG) as a biomarker of endocrine disruption in fish has led to VTG being considered as a potential tool in invertebrates. Among aquatic invertebrate models in ecotoxicology, the copepods are considered as reference species in marine, estuarine and freshwater ecosystems. In this context, we identified a VTG cDNA in Eurytemora affinis. The Ea-VTG2 cDNA is 5416bp in length with an open reading frame (ORF) of 5310bp that encodes a putative protein of 1769 amino acids residues. Phylogenetic analysis confirmed the hypothesis of a VTG duplication event before the emergence of the copepod species. The analysis of the Ea-VTG2 expression by qPCR in males and females according to their reproductive stages allowed transcript basal levels to be determined. The expression pattern revealed a gradual increase of transcript levels during maturation in females. Important inter-sex differences were observed with a VTG level in males ranging from about 1900- to 6800-fold lower than in females depending on their stage. Moreover, the protein was only detected in ovigerous females. The inducibility of Ea-VTG2 by chemicals was studied in males exposed to either a model of endocrine disruptor in vertebrates i.e. 4-nonylphenol (4-NP) or a crustacean hormone i.e. Methyl Farnesoate (MF), and in males sampled from a multi-contaminated estuary. No induction was highlighted. The VTG should not be considered as an appropriate biomarker in E. affinis as previously suggested for other crustaceans.

Shotgun Proteomics Analysis Discards Alkali Labile Phosphate as a Reliable Method To Assess Vitellogenin Levels in Mytilus galloprovincialis.

Vitellogenin, the egg yolk precursor, is a well-known biomarker of endocrine disruption in oviparous vertebrates. In invertebrates, such as bivalves, it has been used in the last 10 years for the same purpose, despite the limited knowledge of invertebrate endocrinology. In bivalves, vitellogenin levels are usually estimated using an indirect technique, alkali labile phosphate (ALP), that assumes that vitellogenin is the most abundant phosphorylated protein in the analyzed tissue. In this study, we applied shotgun proteomics for the identification and quantification of vitellogenin in marine mussel gonads and compared the results with those obtained with the ALP method. The proteomic analysis revealed that vitellogenin is only detected in female gonads with expression levels that are rather variable among female mussels at different stages of gonad development. ALP analysis, on the contrary, detected similar amounts of phosphorylated proteins regardless of sex or gonad development stage. These results show evidence that the ALP method is not providing reliable information about Vtg levels, at least in marine mussel gonads. ALP is not a good proxy to assess Vtg levels in marine mussels, and careful verification of the adequacy of the procedure should be done before ALP is further assumed as a proxy of Vtg in other bivalve mollusks.

Ecological risk assessment in a tropical wetland contaminated with gasoline: Tier 1

ABSTRACTTo assess natural attenuation and the efficiency of remediation actions after more than two years a large accident with gasoline spill contaminated a wetland in a tropical region, an Ecological Risk Assessment based on the Dutch Triad was applied. In total, eight surface water-sampling points were distributed randomly in the affected area and on reference area upstream the contaminated site, with similar ecological characteristics. Risks based on chemical, ecotoxicological, and ecological lines of evidence (LoE) were calculated to integrate the environmental risk indexes. The chemical risk was derived from toxic pressure coefficients based on the total BTEX and naphthalene concentrations. Ecotoxicological LoE based on acute toxicity with Daphnia similis and Aliivibrio fischeri bioassays and chronic toxicity with Desmodesmus subspicatus bioassay contributed to raise uncertainty due to low sensitivity of acute assays. Genotoxicity and endocrine disruption biomarkers of Oreochromis niloticus were used to calculate the Biomarker Stress Index (BSI) assumed as Ecological Risk Index. The integration of the Chemical Risk Index with BSI to estimate the Biological Vulnerability Index allowed a meaningful analysis of the threats to the aquatic ecosystem, thereby supporting managers and decision-makers.

Proteomic response of Macrobrachium rosenbergii hepatopancreas exposed to chlordecone: Identification of endocrine disruption biomarkers?

The present work is the first study investigating the impacts of chlordecone, an organochlorine insecticide, on the proteome of the decapod crustacean Macrobrachium rosenbergii, by gel-free proteomic analysis. The hepatopancreas protein expression variations were analysed in organisms exposed to three environmental relevant concentrations of chlordecone (i.e. 0.2, 2 and 20µg/L). Results revealed that 62 proteins were significantly up- or down-regulated in exposed prawns compared to controls. Most of these proteins are involved in important physiological processes such as ion transport, defense mechanisms and immune system, cytoskeleton dynamics, or protein synthesis and degradation. Moreover, it appears that 6% of the deregulated protein are involved in the endocrine system and in the hormonal control of reproduction or development processes of M. rosenbergii (e.g. vitellogenin, farnesoic acid o-methyltransferase). These results indicate that chlordecone is potentially an endocrine disruptor compound for decapods, as already observed in vertebrates. These protein modifications could lead to disruptions of M. rosenbergii growth and reproduction, and therefore of the fitness population on the long-term. Besides, these disrupted proteins could be suggested as biomarkers of exposure for endocrine disruptions in invertebrates. However, further investigations are needed to complete understanding of action mechanisms of chlordecone on proteome and endocrine system of crustaceans.