Vtg Levels Research Articles

Computational and experimental investigations on the interactions of aryloxy-phenoxy-propionate herbicides to estrogen receptor alpha in zebrafish.

When the amount of pesticide exceeds the self-purification ability of the environment, it will be enriched in the human body through the atmosphere, soil, water circulation, etc., threatening human health. Aryloxy-phenoxy-propionate (APP) herbicides are a class of acetyl-CoA carboxylase (ACCase) inhibitor herbicides, widely used in field-weeding of soybean, cabbage, peanut and other crops. However, due to the water circulation, surface runoff and the agronomic practices such as watering irrigation, APP herbicides have the risk of polluting water and destroying the living environment of aquatic organisms. In this paper, a multistep framework combining homology modeling, molecular docking and molecular dynamic simulations were adopted to explore the interactions between APP herbicides and zebrafish estrogen receptor α (ERα) to investigate the estrogenic activities of the herbicides. The structure of zebrafish ERα was modeled by homology modeling, using the human's estrogen receptor α (PDB ID:2YJA) as the template. Then, eight typical APP herbicides were selected to dock with the zebrafish ERα, and it was determined that there were clear interactions between the herbicides and the receptor. The binding patterns of Quizalofop-P-ethyl (QPE), Clodinafop-propargyl (CP) and Haloxyfop-P (HP) with ERα were further investigated by molecular dynamics and binding free energy calculation. The results showed the van der Waals force and electrostatic force were the main driving forces for maintaining the stability of the complex system. In order to verify the theoretical prediction, an exposed experiment was conducted to study the effects of different concentrations of herbicides on VTG level of zebrafish in vivo and the results were consistent with the computational method. The results of this study revealed the mechanism of the action between APP herbicides and zebrafish estrogen receptors, and also provided ideas for optimizing the herbicides.

Mother-to-embryo vitellogenin transport in a viviparous teleost Xenotoca eiseni

Vitellogenin (Vtg), a yolk nutrient protein that is synthesized in the livers of female animals, and subsequently carried into the ovary, contributes to vitellogenesis in oviparous animals. Thus, Vtg levels are elevated during oogenesis. In contrast, Vtg proteins have been genetically lost in viviparous mammals, thus the yolk protein is not involved in their oogenesis and embryonic development. In this study, we identified Vtg protein in the livers of females during the gestation of the viviparous teleost, Xenotoca eiseni Although vitellogenesis is arrested during gestation, biochemical assays revealed that Vtg protein was present in ovarian tissues and lumen fluid. The Vtg protein was also detected in the trophotaeniae of the intraovarian embryo. Immunoelectron microscopy revealed that Vtg protein is absorbed into intracellular vesicles in the epithelial cells of the trophotaeniae. Furthermore, extraneous Vtg protein injected into the abdominal cavity of a pregnant female was subsequently detected in the trophotaeniae of the intraovarian embryo. Our data suggest that the yolk protein is one of the matrotrophic factors supplied from the mother to the intraovarian embryo during gestation in X. eiseni.

Chronic thiamethoxam exposure impairs the HPG and HPT axes in adult Chinese rare minnow (Gobiocypris rarus): Docking study, hormone levels, histology, and transcriptional responses

Thiamethoxam has emerged as an environmental contaminant detected in aqueous environments, and its endocrine-disrupting effect at chronic exposure in teleosts remains unknown. In the present study, a docking experiment and an in vivo test were integrated to systematically explore the toxic mechanisms of thiamethoxam in fish. Histological analysis, plasma VTG and hormone level (E2, 11-KT, T3 and T4) determinations, and HPG and HPT gene expression quantification were performed after Chinese rare minnow (Gobiocypris rarus) was exposed to thiamethoxam (0, 0.5, 5, and 50 μg/L) for 90 days. According to the docking study, thiamethoxam had different interactions with ERα, AR and TRα via hydrogen bonding. A decrease in body length and plasma T4 was observed in both genders. The histological damage in liver and delayed gonadal development were observed in both genders at 50 μg/L thiamethoxam treatment. In males, the following HPG axis genes were upregulated: gnrh and cyp19b in the brain; vtg and cyp19a in the liver; and cyp17 and cyp19a in the gonad. In females, erɑ in the liver was significantly upregulated with 0.5 μg/L thiamethoxam treatment, and cyp17 in the gonad was upregulated with all treatment. The suppression of cyp19a, gnrh, cyp11a, and ttr was observed at the concentration of 5 μg/L in the female liver. Taken together, the endocrine system of Chinese rare minnow might be disrupted after chronic exposure to thiamethoxam.

Underlying mechanisms of reproductive toxicity caused by multigenerational exposure of 2, bromo-4, 6-dinitroaniline (BDNA) to Zebrafish (Danio rerio) at environmental relevant levels

2-bromo-4, 6-dinitroaniline (BDNA) is a mutagenic aromatic amine involved in the production and degradation of Disperse blue 79, one of the most extensively used brominated azo dyes. In our previous study, a multigenerational exposure of BDNA (0.5, 5, 50 and 500 μg/L) to zebrafish from F0 adult to F2 larvae including a recovery group in F2 larvae was conducted. The effects on apical points observed in individuals and the long-term effects predicted on population were all related to reproduction. In this study, we performed molecular analysis to elucidate the underlying mechanisms of the reproductive toxicity of BDNA. In F1 generation, measurement of vitellogenin and transcription levels of genes associated with hypothalamus-pituitary-gland (HPG) axis, estrogen receptor (ER) and androgen receptor (AR) were conducted. There was a decrease in VTG level in the blood of F1 female fish and transcription of genes related to ER was more affected than that of genes related to AR. These results were consistent with adverse effects that sexual differentiation was biased towards males and fecundity was impaired in a concentration-dependent manner in adults of F1 generation after 150 days exposure. In F2 generation, global gene transcriptions of F2 larvae were investigated. It was uncovered that processes related to apoptosis, development and DNA damage were strongly affected. Alterations to these biological pathways accounted for the irreversible parental influence on a significant decrease in hatchability and increase in abnormality of F2 larvae. All evidence suggested that the multigenerational exposure of BDNA posed lasting effects transmitted from parents to offspring that persisted after exposure ceased.

New methods for purification of Paralichthys olivaceus lipovitellin and immunoassay-based detection of vitellogenin

Increasing levels of estrogenic pollution in marine environments has made the development of reliable biological detection techniques urgently needed. In this study, Japanese flounder (Paralichthys olivaceus) lipovitellin (Lv) was purified and used to establish three immunological methods for the detection of vitellogenin (Vtg), a biomarker for environmental estrogens. Firstly, five different methods were employed to purify Lv, among which water-precipitation was the fastest and easiest way to purify Lv. Japanese flounder Lv was characterized as a phospholipoglycoprotein with a molecular weight of ∼369 kDa. Using purified Lv and its specific polyclonal antibody, a sandwich enzyme-linked immunosorbent assay (ELISA) was developed. This assay had a working range from 7.8 to 250 ng/mL and a detection limit of 3.1 ng/mL. Furthermore, we developed an immunohistochemistry (IHC) and an immunofluorescence (IF) assay, both of which allowed visual detection of liver Vtg. Finally, Vtg induction in plasma and liver of juvenile Japanese flounders exposed to 17β-ethinylestradiol (EE2) was measured using these three methods. Exposure to 10 and 50 ng/L EE2 significantly increased plasma Vtg levels, and obvious positive fluorescence signals were observed near the liver sinusoidal vessels. These results confirmed that the methods developed effectively detected estrogenic activity of exogenous chemicals. Therefore, this study provides reliable methodologies for biomonitoring of estrogenic pollution in marine environments.

Effects of xenostrogen treatment (4-Nonylphenol, Bisphenol A) on vitellogenin expression in juvenile Cyprinus carpio

The aim of this study was to examine some potential effects of 4-Nonylphenol (4-NP), Bisphenol A (BPA), and mixture of them on common carp, Cyprinus carpio and demonstrate induction of vitellogenin (Vtg) as a biomarker for screening aquatic ecosystems. These compounds with other estrogenic chemicals may be responsible for disrupting effects observed in fish. A total of 100 juvenile carp were treated experimentally with 17β-estradiol (E2), and increasing doses of 4-NP, BPA and also mixture of them. Then the plasma Vtg levels were measured using indirect competitive ELISA. Results showed a significant (P<0.05) increase in Vtg levels of exposed carps with E2, maximum 4-NP, BPA and their mixture. The group treated with E2 showed high induction (490%) with respect to controls. The groups treated with maximum dose of 4-NP and BPA showed also induction of Vtg, whereas the group treated with their mixture (3 mg kg−1 b.w. 4-NP and 0.01 mg kg−1 b.w. BPA) showed the highest induction (2126%). Finally, results showed that mixture of 4-NP and BPA exhibited stronger (synergistic) effects. So that, large scale monitoring of estrogenic effects caused by contamination may be considered as a biomarker in urban and industrial effluents.

Effects of PCB-77 in adult zebrafish after exposure during early life stages

Polychlorinated biphenyls (PCBs) are a class of industrial chemicals that cause endocrine changes, since they are able to bind to estrogen receptors and interfere with estrogen-regulated processes, such as fish vitellogenesis. Therefore, the present work aimed to assess potential endocrine effects of PCB-77 exposure during zebrafish (Danio rerio) gonadal differentiation. To achieve that, zebrafish juveniles were exposed to increasing concentrations of PCB-77 for 14 days during a critical window of gonad differentiation (30–44 days post-fertilization). Vitellogenin (Vtg) levels and several endpoints such as survival, growth, gonadosomatic index (GSI) and hepatosomatic index were recorded at the end of exposure and/or after 3 months in clean medium. The results obtained showed a reduction of Vtg-like proteins in juveniles, just after exposure to PCB-77 accompanied, after 3 months, by a decrease in gonadal Vtg levels and GSI of females. These results suggest that exposure to PCB-77 during the critical window of gonadal differentiation decreased vitellogenesis in juvenile zebrafish which lasted until adulthood affecting the normal development of female gonad, which might have further implications in reproduction success.

Synergistic effects of estradiol and 11-ketotestosterone on vitellogenin physiology in the shortfinned eel (Anguilla australis).

Estradiol-17β (E2) and 11-ketotestosterone (11KT) have been implicated in vitellogenesis and in regulating expression of the follicle-stimulating hormone receptor (fshr), respectively. To override the captivity-induced reproductive block in shortfinned eel, Anguilla australis, we hypothesized that in combination, 11KT and E2 would stimulate ovarian uptake of vitellogenin (Vtg). Early pubertal eels received hormone implants containing varying concentrations of E2 (0, 0.2, 2, 5 mg) with or without 11KT (1 mg). Vtg levels were determined in plasma, liver, and ovarian tissues by histological examination, qPCR, immunoblotting, or single radial immunodiffusion. The expression of gonadotropin-beta subunits and gonadotropin receptors in the pituitary and ovary, respectively, were analyzed to determine mechanisms by which steroid effects may be exerted. When administered alone, E2 increased hepatic production and plasma levels of Vtg. In contrast, 11KT decreased plasma levels of Vtg, seemingly reducing its production. Neither 11KT nor E2 could induce uptake of Vtg into oocytes, although E2 treatment appeared necessary for uptake to occur. This was the case despite 11KT dramatically increasing both oocyte size and fshr mRNA levels. Astonishingly, the uptake of Vtg was successfully induced by co-treatment with 11KT and E2, suggesting that 11KT might facilitate the incorporation of Vtg into the developing oocyte. These results highlight the potential of sex steroid co-treatment, an approach aimed at mimicking oogenesis in wild eels, to induce vitellogenesis, specifically ovarian yolk deposition, even in the absence of exogenous gonadotropin treatment.

Evaluation of the estrogen receptor alpha as a possible target of bifenthrin effects in the estrogenic and dopaminergic signaling pathways in zebrafish embryos

Bifenthrin (BF) is a pyrethroid insecticide widely used in urban and agricultural applications. Previous studies in embryos of zebrafish have shown that BF can affect estradiol biosynthesis and the dopaminergic system. To examine the role of the estrogen receptor (ER) in the endocrine effects of BF, embryos were exposed for 96 h to a mixture of 0.15 and 1.5 μg/L BF and an ER agonist (17α-ethynylestradiol – EE2) at 0.09 μg/L. Transcripts related to estrogenic (vitellogenin VTG) and dopaminergic (tyrosine hydroxylase (TH), dopamine receptor 1 (DR1), monoamine oxidase (MAO), and catechol-O-methyltransferase b (COMTb)) signaling pathways were investigated by qRT-PCR. Dopamine (DA) and its metabolites (homovanillic acid (HVA) and 3,4-dihydroxyphenylacetic acid (DOPAC)) were also measured. There was a significant increase in VTG, DR1, MAO and COMTb mRNA levels and HVA-DA ratios within all zebrafish embryos exposed to EE2, including EE2 alone, 0.15 μg/L BF + EE2 and 1.5 μg/L BF + EE2. A significant decrease in homogenate concentrations of DA was observed within all zebrafish embryos exposed to EE2, which included EE2 alone, 0.15 μg/L BF + EE2 and 1.5 μg/L BF + EE2. Co-exposure of BF with EE2 failed to diminish estrogenic or dopaminergic signaling in embryos. Additionally, embryos with diminished ERα expression by morpholino injection were exposed to 0.15 μg/L BF, 1.5 μg/L BF and 0.09 μg/L EE2, with subsequent gene expression measurements. ERα knockdown did not prevent the effects of BF, indicating ERα may have a limited role in the estrogenic and dopaminergic effects caused by BF in zebrafish embryos.

Plasma biomarkers in juvenile marine fish provide evidence for endocrine modulation potential of organotin compounds

Organotin compounds, such as tributyltin (TBT) and triphenyltin (TPT), have been widely used to control marine fouling. Here, we show that organotin stimulation reduces the hormone levels in the plasma of two economically important aquaculture fish. Blood plasma samples were collected from juvenile red seabream and black rockfish exposed to environmentally realistic concentrations of TBT and TPT for 14 days. The levels of two plasma biomarkers, namely the yolk protein precursor vitellogenin (VTG) and the sex steroid 17β-estradiol (E2), were measured to determine the endocrine disrupting potential of the organotin compounds. Both organotin compounds were dose-dependently accumulated in the blood of two fish. Exposure to waterborne TBT and TBT significantly decreased the plasma VTG levels in both the juvenile fish in a dose-dependent manner. In contrast, the treatment with E2, a well-known VTG inducer, significantly increased the plasma VTG levels in both the fish. In addition, the mRNA levels of vtg were also downregulated in the liver tissues of both the fish at 100 and/or 1000 ng L−1 of TBT or TPT exposure. The plasma E2 titers were significantly suppressed at 100 and/or 1000 ng L−1 of TBT or TPT exposure for 14 days compared to their titer in the control. Since estrogen directly regulates vtg gene expression and VTG synthesis, our results reveal the endocrine disrupting potential of organotin compounds, and subsequently the endocrine modulation at early stage of fish can trigger further fluctuations in sexual differentiation, maturation, sex ration or egg production. In addition, the results demonstrate their effects on non-target organisms, particularly on animals reared in aquaculture and fisheries.

Synergistic potential of fenvalerate and triadimefon on endocrine disruption and oxidative stress during rare minnow embryo development.

Pyrethroids have been reported to interact synergistically when co-exposed with azoles fungicides in different organisms. In the present study, we investigated the mixture toxicity of fenvalerate (FEN) and triadimefon (TDF) toward embryos of Gobiocypris rarus after 96 h exposure. Results demonstrated that TDF enhanced the acute toxicity of FEN. Exposure to binary mixtures of FEN and TDF resulted in synergistic responses of endocrine disruption by inducing the transcripts of several genes including vtg, erα, erβ1, erβ2, cyp19a, cyp1a, cyp4, cyp11a, gnrh3, gnrhr1a, star, and dmrt1. Furthermore, FEN and TDF mixture increased the VTG level and aromatase activity in rare minnow embryos. FEN and TDF co-exposure also regulated the mRNA of vezf, hsp70, p53, gadd45α, induced the synthesis of ROS and activity of GST, suggesting the synergistic potential of oxidative stress induced by FEN and TDF co-exposure. The results indicated that binary mixtures of FEN and TDF could simultaneously induce endocrine disruption and oxidative stress in a synergistic manner during rare minnow embryo development.

Biological response of zebrafish after short-term exposure to azoxystrobin

Azoxystrobin (AZ) is a broad-spectrum systemic fungicide that widely used in the world. The present study investigated the toxicity effects on zebrafish after short-term exposure of AZ. Results demonstrated that the larval stage was most susceptible to AZ in the multiple life stages of zebrafish, with 96 h-LC50 value of 0.777 mg/L. Zebrafish larvae were exposed to different AZ concentrations (0, 0.1, 1, 10, 100 μg/L) and examined on 24, 48 and 72 h. It was found that AZ induced ROS accumulation, increased GST, GPX and POD activity and the transcriptions of antioxidant and stress response related genes, while the opposite trend occurred for SOD and CAT activity in 24-h or 48-h exposure period. The increased E2 and VTG levels in zebrafish larvae, and altered transcription levels of regulatory and steroidogenic genes in the hypothalamus-pituitary-gonad (HPG) axis indicated the endocrine disruption capacity of AZ. The transcripts of mdm2, p53, ogg1, bcl2, bbc3, cas8 and cas9 involved in cell apoptosis, and the mRNA levels of cytokines and chemokines such as cxcl-c1c, ccl, il-1β, il-8, ifn, and tnfα were in accordance with the trends of the examined genes involved in oxidative stress and endocrine system. The results suggested that short-term exposure to AZ might impose ecotoxicological effects on zebrafish larvae, and the information presented here also provide molecular strategies and increase mechanistic understanding of AZ-induced toxic response, and help elucidate the environmental risks of AZ.

Effects of triclosan (TCS) on hormonal balance and genes of hypothalamus-pituitary- gonad axis of juvenile male Yellow River carp (Cyprinus carpio)

Triclosan (TCS) is a broad spectrum antimicrobial agent which has been widely dispersed and determinated in the aquatic environment. However, the effects of TCS on reproductive endocrine in male fish are poorly understood. In this study, male Yellow River carp (Cyprinus carpio) were exposed to 0, 1/5, 1/10 and 1/20 LC50 (96 h LC50 of TCS to carp) TCS under semi-static conditions for 42 d. Vitellogenin (Vtg), 17β-estradiol (E2), testosterone(T), gonadotropin (GtH), and gonadotropin-releasing hormone (GnRH) levels were measured by enzyme-linked immunosorbent assay (ELISA). Meanwhile, we also examined the mRNA expressions of aromatase, GtHs-β, GnRH, estrogen receptor (Er), and androgen receptor (Ar) by quantitative Real-time Polymerase Chain Reaction (qRT-PCR). TCS induced Vtg levels of hepatopancreas, E2 levels of serum, and inhibited Ar and Er mRNA levels, suggesting that the induction of Vtg production by TCS was indirectly caused by non-Er pathways. TCS-induced Vtg levels by interfering with the reproductive axis at plenty of latent loci of male carps: (a) TCS exposure increased the aromatase mRNA expression of hypothalamus and gonad aromatase, consequently increasing serum concentrations of E2 to induce Vtg in hepatopancreas; (b) TCS treatment changed GtH-β and GnRH mRNA expression and secretion, causing the disturbance of reproductive endocrine; (c) TCS exposure decreased Ar mRNA levels, indicating potential Ar-mediated antiandrogen action. These mechanisms showed that TCS may induce Vtg production in male carp by non-Er-mediated pathways.

Levels of 17β estradiol, vitellogenin, and prostaglandins during the reproductive cycle of Oreochromis niloticus

Circulating levels of estradiol, vitellogenin and prostaglandins could be references of oocytes quality in the aquaculture industry. This study evaluated the performance of 17β-estradiol (E2) and vitellogenin (Vtg) levels in plasma and prostaglandins in oocytes (PGE 2) during the reproductive period of Oreochromis niloticus in a commercial farm in Ecuador. Adults fish females were treated with estradiol-synthetic (ES) and tilapia pituitary extracts (TP) during 25 days. For quantification of E2 and PGE 2, an enzyme-linked immunosorbent assay (ELISA) was used, while for Vtg relative quantification a Western blot analysis was performed. Circulating E2 concentrations were different during the first week of follow-up ( P < 0.05) resulting in different levels of Vtg per treatment ( P < 0.05) affecting on gonadal growth. The ovaries had an asynchronous development, where the mature oocytes proportions were higher with ES and TP than control. Relative fecundity was different between treatments ( P < 0.05), where TP generated 15.7 ± 5.8 oocytes per g of spawning female. Furthermore, concentrations of PGE 2 were different between treatments ( P < 0.05). The information reported in this study will be useful to improve the reproduction methods and have some biological signals before ovulation in tilapia commercial culture.

Effects of 2,2',4,4'-tetrabromodipheny ether (BDE-47) on gonadogenesis of the manila clam Ruditapes philippinarum

The reported adverse effects of Polybrominated diphenyl ether (PBDE) congeners on gonadogenesis in fish may also occur in marine bivalves especially the burrowing species such as manila clam Ruditapes philippinarum. In this study, clams were exposed to BDE-47 for 25days at 0, 0.1 and 1μg/L. By using the water temperature control method, gonadal maturation from resting to ripe stage were observed successively in both the control and the treatment groups during 25days. The results showed that exposure to BDE-47 at concentration below 1μg/L did not delay the gonadogenesis process of the clam R. philippinarum, and no evidence of adverse effects of BDE-47 on clam gonadal histology was observed. However, exposure to 1μg/L BDE-47 caused significant decreases of haemolymph testosterone levels in both female and male clams at day 5 and day 15. The mRNA expression of 3β-HSD in females exposed to BDE-47 was significantly decreased at day 5, while mRNA expression of 17β-HSD and CYP17 was not significantly changed in either sex. Exposure to BDE-47 also resulted in up-regulation of the mRNA expression of vitellogenin (Vtg) in both sexes and spermatogenesis associated protein 4-homolog (SAP4) in males. These results suggest a potential contribution of BDE-47 to reproductive disruption in the manila clams, especially in males. This study demonstrates the promising utility of water temperature control method in conjunction with histological endpoints and biomarkers such as mRNA levels of Vtg in determining the reproductive disturbances caused by EDCs on bivalves.

Tools to minimize interlaboratory variability in vitellogenin gene expression monitoring programs.

The egg yolk precursor protein vitellogenin is widely used as a biomarker of estrogen exposure in male fish. However, standardized methodology is lacking and little is known regarding the reproducibility of results among laboratories using different equipment, reagents, protocols, and data analysis programs. To address this data gap we tested the reproducibility across laboratories to evaluate vitellogenin gene (vtg) expression and assessed the value of using a freely available software data analysis program. Samples collected from studies of male fathead minnows (Pimephales promelas) exposed to 17α-ethinylestradiol (EE2) and minnows exposed to processed wastewater effluent were evaluated for vtg expression in 4 laboratories. Our results indicate reasonable consistency among laboratories if the free software for expression analysis LinRegPCR is used, with 3 of 4 laboratories detecting vtg in fish exposed to 5 ng/L EE2 (n = 5). All 4 laboratories detected significantly increased vtg levels in 15 male fish exposed to wastewater effluent compared with 15 male fish held in a control stream. Finally, we were able to determine that the source of high interlaboratory variability from complementary deoxyribonucleic acid (cDNA) to quantitative polymerase chain reaction (qPCR) analyses was the expression analysis software unique to each real-time qPCR machine. We successfully eliminated the interlaboratory variability by reanalyzing raw fluorescence data with independent freeware, which yielded cycle thresholds and polymerase chain reaction (PCR) efficiencies that calculated results independently of proprietary software. Our results suggest that laboratories engaged in monitoring programs should validate their PCR protocols and analyze their gene expression data following the guidelines established in the present study for all gene expression biomarkers. Environ Toxicol Chem 2017;36:3102-3107. Published 2017 Wiley Periodicals Inc. on behalf of SETAC. This article is a US government work and, as such, is in the public domain in the United States of America.

Gonadal intersex in smallmouth bass Micropterus dolomieu from northern Indiana with correlations to molecular biomarkers and anthropogenic chemicals

Over the past decade, studies have shown that exposure to endocrine disrupting chemicals (EDCs) can cause gonadal intersex in fish. Smallmouth bass (Micropterus dolomieu) males appear to be highly susceptible to developing testicular oocytes (TO), the most prevalent form of gonadal intersex, as observed in various areas across the U.S. In this study, prevalence and severity of TO was quantified for smallmouth bass sampled from the St. Joseph River in northern Indiana, intersex biomarkers were developed, and association between TO prevalence and organic contaminants were explored. At some sites, TO prevalence reached maximum levels before decreasing significantly after the spawning season. We examined the relationship between TO presence and expression of gonadal and liver genes involved in sex differentiation and reproductive functions (esr1, esr2, foxl2, fshr, star, lhr and vtg). We found that vitellogenin (vtg) transcript levels were significantly higher in the liver of males with TO, but only when sampled during the spawning season. Further, we identified a positive correlation between plasma VTG levels and vtg transcript levels, suggesting its use as a non-destructive biomarker of TO in this species. Finally, we evaluated 43 contaminants in surface water at representative sites using passive sampling to look for contaminants with possible links to the observed TO prevalence. No quantifiable levels of estrogens or other commonly agreed upon EDCs such as the bisphenols were observed in our contaminant assessment; however, we did find high levels of herbicides as well as consistent quantifiable levels of PFOS, PFOA, and triclosan in the watershed where high TO prevalence was exhibited. Our findings suggest that the observed TO prevalence may be the result of exposures to mixtures of nonsteroidal EDCs.

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.

Reproductive effects of oestrogenic endocrine disrupting chemicals in Astyanax rivularis inhabiting headwaters of the Velhas River, Brazil

The Velhas River is the most polluted river in the state of Minas Gerais, south-eastern Brazil. Due to its historical and environmental relevance, the aim of this study was to evaluate the effects of oestrogenic endocrine disruptors on the reproduction of the lambari Astyanax rivularis, a small-sized species found in headwaters of the São Francisco River basin. Quarterly field samplings were carried out during a reproductive cycle in three streams of the upper Velhas River: S1 (reference site) and S2 and S3 (sites contaminated by untreated sewage). The main oestrogenic compounds were evaluated in water using HPLC/MS. Molecular, histological and reproductive biomarkers were assessed in liver and gonad. The results showed higher average concentrations of oestradiol (>200ng/l) in S2 and S3, oestrone (>250ng/l) in S2 as well as oestriol (>200ng/l), bisphenol A (>190ng/l), and nonylphenol (>600ng/l) in S3 compared to S1 (<70ng/l for all compounds). In S2 and S3, there was an increase in the proportion of females, higher ELISA levels of vitellogenin (Vtg) and proteins of the zona radiata (Zrp) in liver males. Insulin-like growth factor (IGF-I) levels were lower in S2 males, which also had a smaller body size, a smaller seminiferous tubule diameter, a higher proportion of spermatogonia, and lower proportion of spermatozoa in relation to S1. Histopathological analyses detected an increase in yolk deficient oocytes and over-ripening in the contaminated sites, and these alterations were associated to a reduction of hepatic Vtg levels and a delay in spawning, respectively. Intersex specimens with perinucleolar follicles in a multifocal distribution in the testis were detected in S2 and S3. These results indicate that chronic exposure to oestrogenic compounds induced endocrine disruption that may affect wild populations of A. rivularis in the Velhas River.

Evaluating the potential of effluent extracts from pulp and paper mills in Canada, Brazil, and New Zealand to affect fish reproduction: Estrogenic effects in fish.

The authors examined the potential of pulp mill effluent from pulp-producing countries (Canada, Brazil, New Zealand) to affect fish reproduction. Specifically, the estrogenic effects in juvenile rainbow trout (Oncorhynchus mykiss) pulse-exposed to 11 different mill effluent extracts (intraperitoneal injections of solid-phase extraction-dichloromethane nonpolar fraction). The results indicated that effluent extracts were estrogenic in juvenile trout irrespective of the gender, as reflected by increasing level of plasma vitellogenin (VTG; Brazil > New Zealand > Canada). Despite the high variability observed among mills, differences in VTG levels were related to the type of mill process (kraft > elementary chlorine-free kraft > thermomechanical pulping). Moreover, effluent treatments did not appear to significantly decrease VTG induction. A consistent estrogenic effect was observed in those mills that process a combination of feedstocks (softwood and hardwood), with the highest increase in VTG related to eucalyptus feedstock. The results demonstrate significant estrogenic effects of pulp mill effluents on chronically exposed juvenile trout, suggesting that in vivo metabolic activation of precursors is necessary to cause the observed increases in VTG levels. This molecular estrogenic response provides a useful starting point for predicting population-level impacts through the adverse outcome pathway methodology. Environ Toxicol Chem 2017;36:1547-1555. © 2016 SETAC.