Vtg Production Research Articles

Endocrine disruptive toxicity of cypermethrin in Labeo catla: Involvement of genes and proteins related to the HPG axis

Cypermethrin (CYP) is a synthetic pyrethroid abundantly used in agriculture and aquaculture. It is an established potent endocrine disruptor to fish, yet the molecular mechanism behind its reproductive toxicity remains unclear. In this study, fish Labeo catla (Catla) was exposed to environmentally relevant concentration of CYP (0.7 μg/L) and 0.14 μg/L for 30 days. The changes in circulating sex steroids, genes, and hormones linked to the hypothalamic-pituitary-gonadal (HPG) axis, stress response and associated histological alterations were studied. Significant decline (P < 0.05) in serum 17 beta (β) estradiol (E2), 11 ketotestosterone (11-KT), and brain (FSH and GnRH) were observed in 0.7 μg/L dose of CYP. These effects may be due to the down-regulated expression of the upstream genes of the HPG axis i.e. Kiss 1 and Kiss 2, which further downregulates the expression of the GnRH gene. The decreased level of E2 and 11-KT also affects the vitellogenin (Vtg) gene expression, reducing the production of Vtg, a crucial protein for ovarian development. Principal component analysis (PCA) revealed the relationship between CYP and the biosynthesis of sex steroids. The toxic effect of CYP was also visible in antioxidant enzyme assay and related histological alterations. Overall, the study elucidated that long-term exposure to CYP, even at an environmentally relevant dose, may affect reproductive potential and fish recruitment. The study provides important insights into molecular mechanisms underlying CYP-induced endocrine disruption in fish, and it also raises questions about CYP's potential toxicity at environmentally relevant concentration in terms of understanding ecological risk.

Fusion and expansion of vitellogenin vesicles during Caenorhabditis elegans intestinal senescence.

Some of the most conspicuous aging phenotypes of C.elegans are related to post-reproductive production of vitellogenins (Vtg), which form yolk protein (YP) complexes after processing and lipid loading. Vtg/YP levels show huge increases with age, and inhibition of this extends lifespan, but how subcellular and organism-wide distribution of these proteins changes with age has not been systematically explored. Here, this has been done to understand how vitellogenesis promotes aging. The age-associated changes of intestinal vitellogenin vesicles (VVs), pseudocoelomic yolk patches (PYPs), and gonadal yolk organelles (YOs) have been characterized by immuno-electron microscopy. We find that from reproductive adult day 2 (AD 2) to post-reproductive AD 6 and AD 9, intestinal VVs expand from 0.2 to 3-4μm in diameter or by >3000 times in volume, PYPs increase by >3 times in YP concentration and volume, while YOs in oocytes shrink slightly from 0.5 to 0.4μm in diameter or by 49% in volume. In AD 6 and AD 9 worms, mislocalized YOs found in the hypodermis, uterine cells, and the somatic gonadal sheath can reach a size of 10μm across in the former two tissues. This remarkable size increase of VVs and that of mislocalized YOs in post-reproductive worms are accompanied by extensive fusion between these Vtg/YP-containing vesicular structures in somatic cells. In contrast, no fusion is seen between YOs in oocytes. We propose that in addition to the continued production of Vtg, excessive fusion between VVs and mislocalized YOs in the soma worsen the aging pathologies seen in C.elegans.

Insights into the development of hepatocellular fibrillar inclusions in European flounder (Platichthys flesus) from UK estuaries

Hepatocellular fibrillar inclusions (HFI) are an unusual pathology of unknown aetiology affecting European flounder (Platichthys flesus), particularly from estuaries historically impacted by pollution. This study demonstrated that the HFI prevalence range was 6–77% at several UK estuaries, with Spearman rank correlation analysis showing a correlation between HFI prevalence and sediment concentrations of ∑PBDEs and ∑HBCDs. The data showed that males exhibit higher HFI prevalence than females, with severity being more pronounced in estuaries exhibiting higher prevalence. HFI were not age associated indicating a subacute condition. Electron microscopy confirmed that HFI were modified proliferating rough endoplasmic reticulum (RER), whilst immunohistochemistry provided evidence of VTG production in HFI of male P. flesus. Despite positive labelling of aberrant VTG production, we could not provide additional evidence of xenoestrogen exposure. Gene transcripts (VTG/CHR) and plasma VTG concentrations (>1 μg ml−1), were only considered elevated in four male fish showing no correlation with HFI severity. Further analysis revealed that reproductively mature female P. flesus i.e. >3-year-old, did not exhibit HFI, whereas males of all ages were affected. This, combined with previous reports that estradiol (E2) can impair mixed function oxygenase activity, supports a hypothesis that harmful chemical metabolites (following phase 1 metabolism of their parent compounds) are potentially responsible for HFIs observed in male and ≤ 3-year-old female fish. Consequently, HFI and xenoestrogenic induced VTG production could be independent of each other resulting from different concurrent toxicopathic mechanisms, although laboratory exposures will likely be the only way to determine the true aetiology of HFI.

Effects of a chronic exposure to different water temperatures and/or to an environmental cadmium concentration on the reproduction of the threespine stickleback (Gasterosteus aculeatus)

Knowledge about combined effects of chemicals and temperature on reproductive capacity of fish are rare in literature, especially when it comes to the effects of chronic low-dose chemical exposure combined to the thermal stress. The aim of the study was to evaluate the single and combined effects of temperature (16, 18, 21 °C) and an environmentally relevant concentration of waterborne cadmium (1 µg L−1, nominal concentration) on the reproductive outputs of threespine stickleback (Gasterosteus aculeatus), and their consequences on offspring survival parameters. The high temperature (21 °C) was the only factor that affected parental parameters (gonadosomatic index “GSI”, and vitellogenin “VTG” particularly). On females, 21 °C had a stimulating effect on gonadal development evaluated by an early increase, followed by a sharp decrease of GSI, probably indicating gonadal atresia. Promoting effect of temperature was corroborated by an early production of VTG. In vitro fertilization assays showed interesting results, particularly cadmium effects. As it was supposed, high temperature had a negative impact on offspring parameters (significant decrease in survival and an increase of unhatched embryos). Parental exposure to the very low concentration of cadmium had also negative consequences on mortality rate (significant increase) and hatching rate (significant decrease). Our results indicate that in a global warming context, high temperature and its combination with contaminant may impact reproductive capacity of G. aculeatus, by decreasing parental investment (low eggs and/or sperm quality).

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.

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.

Effects of triclosan on hormones and reproductive axis in female Yellow River carp (Cyprinus carpio): Potential mechanisms underlying estrogen effect

Triclosan (TCS), a member of the class of compounds called pharmaceutical and personal care products (PPCPs), is a broad antibacterial and antifungal agent found in a lot of consumer products. However, TCS hormone effect mechanism in teleost female fish is not clear. Female Yellow River carp (Cyprinus carpio) were exposed to 1/20, 1/10 and 1/5 LC50 TCS (96h LC50 of TCS to carp) under semi-static conditions for 42days. Vitellogenin (Vtg), 17β-estradiol (E2), testosterone(T), estrogen receptor (Er), 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, and Er by quantitative real-time PCR (qRT-PCR). The results indicated that 1/5 LC50 TCS induced Vtg in hepatopancreas of female carps by interference with the hypothalamic–pituitary–gonadal (HPG) axis at multiple potential loci through three mechanisms: (a) TCS exposure enhanced the mRNA expression of hypothalamus and gonadal aromatase which converts androgens into estrogens, subsequently increasing serum concentrations of E2 to induce Vtg in hepatopancreas; (b) TCS treatment increased GnRH and GtH-β mRNA expression and secretion, causing the disturbance of reproductive endocrine and the increase of E2 to induce Vtg in hepatopancreas; (c) TCS exposure enhanced synthesis and secretion of Er, then it bound to Er to active Vtg synthesis. These mechanisms showed that TCS may induce Vtg production in female Yellow River carp by Er-mediated and non-Er-mediated pathways.

Molecular Characteristic of Giant Grouper (Epinephelus Lanceolatus)Vitellogenin

The Vitellogenin (Vtg) gene sequence acts as an indicator to the fish reproduction, which can adapt to the environment factor or can influence the gonad development. The Vtg nucleotide sequence from Giant grouper was characterized using bioinformatics software. A homology search of the deduced amino acid sequence of the obtained Vtg DNAs (compare with 13 species) was carried out using the National Centre for Biotechnology Information website. Clustal W analysis was constructed a phylogenetic tree by using Molecular Evolutionary Genetic Analysis MEGA version 5.2.2. In order to verify the Vtg gene sequences obtaind and elucidate structure-function relationship in Vtg, by using DELTA BLAST of 3-D structure of Giant grouper with others fishes. Result of phylogenetic analysis using Maximum Likelihood (ML) and Neighbour Joining (NJ) showed tree analysis generated two separated tree topology. This similarity (0.015 distance matric viewer) was closely related in terms of their Vtg gene sequence although from different environmental and ecological conditions. In general, showed that Epinephelus lanceolatus Vtg is evolutionary more related to Poecilia latipinna. Epinephelus lanceolatus shows four main domains (Vitellogenin-N, DUF1943, DUF1944 and VMD), similarly found in Dicentrachus labrax but was different compare to Clarias macrochepalus, Catla catla, and Danio rerio. This indicates characteristic of Vtg domain for freshwater species is control by present of VMD in Vtg. The molecular approach can be done on Giant Grouper to understand the molecular respond towards fish growth and determine the individual of Giant grouper that has potential to increasing the Vtg production for increasing eggs quality.

Evaluation of the sensitivity, responsiveness and reproducibility of primary rainbow trout hepatocyte vitellogenin expression as a screening assay for estrogen mimics

Vitellogenin (Vtg) induction in primary fish hepatocytes has been proposed as an in vitro screening assay for ER agonists and antagonists, but has not yet been used extensively as a high-throughput screening tool due to poor reproducibility, sensitivity and overall feasibility. The present work has evaluated the role of seasonal variation, normalization, optimal culture and assay conditions on the sensitivity, responsiveness and reproducibility of in vitro vtg gene mRNA and protein expression in rainbow trout (Oncorhynchus mykiss) primary hepatocytes using the xenoestrogen 17α-ethynylestradiol (EE2) as a test chemical. The results show that primary hepatocytes display a relatively high individual and seasonal variation in both Vtg mRNA and protein induction potential, although less variance was observed in assay sensitivity. Data normalization of assay response to maximum (3nM EE2) and minimum (DMSO) Vtg production dramatically reduced this variance and led to improved assay reproducibility. A time-dependent response was observed both for mRNA and protein expression, reaching maximum Vtg induction after 96h of exposure, although reproducible concentration response curves for both Vtg mRNA and protein could be obtained already after 48h. A need for chemical re-exposure of the hepatocytes was identified to be important for sustaining exposure concentrations in extended studies (>48h), whereas different plate formats (96, 24 or 6 wells) did not affect the bioassay performance. In conclusion, standardization of hepatocyte bioassay and test conditions as well as data-normalization procedures are proposed to be instrumental for more consistent and comparable results in future use of this in vitro assay.

Endocrine disruption effects of p,p'-DDE on juvenile zebrafish.

The persistent organic pollutant p,p'-DDE, the major metabolite of the insecticide DDT, has displayed evidence of endocrine disruption through the inhibition of androgen binding to androgen receptors in different species. Although p,p'-DDE was continuously detected in wild fish with abnormal gonad development such as intersex, little is known about its mode of action during gonad development in fish. To elucidate the potential endocrine effects of this pollutant in zebrafish (Danio rerio), juveniles (30 days post hatch) were exposed to p,p'-DDE during the critical window of sexual differentiation. Fish were exposed to sublethal concentrations ranging from 0.01 to 20 µg l(-1) over 14 days and were maintained in control water for an additional 4 months. As core endpoints, the vitellogenin (vtg) concentration was measured at the end of exposure, and sex ratio and the gonadosomatic index were assessed 4 months after the end of exposure. An increase in vtg production in whole body homogenate was observed in fish exposed to 0.2 and 2.0 µg l(-1) p,p'-DDE. No significant differences were displayed in morphological parameters such as the gonadosomatic index of males and females or sex ratio. However, exposed females presented histopathological changes that include the reduction of the number of mature oocytes, which might impair their successful reproduction. These results demonstrate the ability of p,p'-DDE to cause endocrine disruption in zebrafish exposed during gonad differentiation of juvenile specimens. Furthermore, vtg induction by p,p'-DDE in juvenile zebrafish arises as a predictive marker for adverse effects of this DDT metabolite on the ovarian function of female zebrafish.

In vitroeffect of pituitary, interrenal and gonadal hormones on vitellogenin synthesis in primary hepatocyte cultures ofChalcalburnus tarichi

Summary Vitellogenin (Vtg) is an important precursor yolk protein in egg-laying vertebrates, including fish. The 17β-oestradiol (E2) plays a crucial role in the Vtg synthesis; moreover, certain hormones can stimulate Vtg synthesis. We investigated the possible role of E2, carp recombinant growth hormone (crGH), insulin (Ins), progesterone (P4) and 11-deoxycortisol (11-DOC) hormones in Vtg synthesis on primary juvenile Chalcalburnus tarichi hepatocyte culture. The amount of Vtg in the medium was measured at 2-day intervals using enzyme-linked immunosorbent assay (ELISA). The hepatocytes were maintained in culture for more than 2 weeks without the addition of serum components. Vitellogenin localization was visualized with the immunofluorescence method in E2-supplemented hepatocytes. Among hormones applied to the culture, only E2 had an influence on Vtg synthesis in a time-dependent manner, while crGH, Ins, P4 and 11-DOC had no effect. However, in hepatocytes stimulated with E2 in combination with P4, a lower Vtg production was seen compared with Vtg produced when hepatocytes were stimulated with E2 alone. P4 proved to have potentiating effects on co-treatment with E2-induced Vtg production. As a result, E2 and P4 are the most important hormones for Vtg synthesis in juvenile C. tarichi hepatocyte culture.

Effects of chronic exposures to the herbicides atrazine and glyphosate to larvae of the threespine stickleback (Gasterosteus aculeatus)

Atrazine (ATR) and glyphosate (GLY) are among the most widely used herbicides in Canada, yet there is relatively little information concerning their toxicity to early life stages of marine fish. The threespine stickleback (Gasterosteus aculeatus) reproduces in coastal habitats which receive runoff of pesticides during the summer, the peak season of herbicide use. Sticklebacks have biomarkers for effects of both estrogenic and androgenic contaminants. Stickleback adults from a clean reference site were allowed to reproduce in the laboratory and the fertilized eggs were incubated until hatching. Larval sticklebacks (<24h old) were exposed for 42d to four concentrations (0.1, 1, 10 and 100μg/l) of either ATR or GLY, a seawater control, a carrier (acetone) control and positive controls for estrogenic (0.05μg/l ethinylestradiol, EE2) and androgenic (3μg/l dihydrotestosterone, DHT) effects. The survivors were measured (length, wet weight) then conserved for biochemical (vitellogenin, VTG, and the male nest-protein spiggin, SPG) and histological (phenotypic sex determination) analyses. There were no significant effects of ATR and GLY exposures on larval survival or growth. Exposure to 3μg DHT/l resulted in a significant effect on growth (body lengths) but did not induce SPG, possibly because of DHT degradation after the 24h solution renewal. VTG was induced after the EE2 exposure, yet neither ATR nor GLY induced production of VTG and SPG. The proportion of mixed sex individuals was higher in the positive controls compared to the negative controls. A single mixed sex individual was found in the group exposed to the lowest dose of atrazine and none in glyphosate expositions. We conclude that these herbicides do not show estrogenic or androgenic effects to early life stages of sticklebacks at environmentally realistic concentrations.

Identification, purification, and immunoassay of stone flounder (Kareius bicolouratus) vitellogenin

Vitellogenin (Vtg), the precursor of egg yolk proteins, serves as nutrients for the developing embryos. Vtg production in male fish has become an excellent biomarker for environmental estrogens. Thus, Vtg synthesis was induced by injection of 17β-estradiol (E2) in stone flounder (Kareius bicolouratus). Plasma from uninduced and E2-treated fish was subjected to native polyacrylamide gel electrophoresis (PAGE) and the gels were stained with Coomassie brilliant blue R250 for proteins, methyl green for phosphates, Sudan black B for lipids, and periodic acid-Schiff’s reagent for carbohydrates. These analyses verified the presence of two phospholipoglycoproteins in the plasma from E2-treated stone flounder, which were tentatively identified as Vtg. Vtg was then isolated by two purification procedures: gel filtration followed by anion exchange chromatography, or selective precipitation combined with anion exchange chromatography. The latter procedure was more effective at isolating Vtg with a high degree of purity. In native PAGE, the apparent molecular masses of Vtgs were determined to be 520 and 550 kDa, respectively. Two major bands with approximate molecular masses of 165 and 106 kDa together with two additional faint bands of lower molecular weights were observed after sodium dodecyl sulfate- PAGE under reducing conditions. These bands exhibited differential affinity for polyclonal antisera against red drum (Sciaenops ocellatus) Vtg in Western blot. The present study established the purification procedure and characterized the electrophoretic and immunological properties of stone flounder Vtg.

Combined effects of oestrogen receptor antagonists on in vitro vitellogenesis

Some environmental compounds are known to have anti-oestrogenic activity and their modes of action (MoA) are believed to include competitive inhibition of 17β-estradiol (E2) binding to the oestrogen receptor (ER) or interference with ER-dependent processes. The presence of multiple compounds having the same MoA may cause concern, as exposure to multiple compounds at concentrations below their threshold for effect can interact with cellular targets to cause effects in combination. The combined effect of mixtures can be assessed using prediction models such as concentration addition (CA) and independent action (IA). The objective of the present study was to determine if the CA and IA prediction models could accurately characterise the combined effects of mixtures of ER antagonists in rainbow trout (Oncorhynchus mykiss) hepatocytes using the ER-mediated production of the oestrogenic biomarker vitellogenin (Vtg) as a screening assay. Model anti-oestrogens (4-hydroxytamoxifen and ZM 189.154) and environmentally relevant compounds (PCBs and PAHs) were tested to ensure inclusion of compounds from different chemical classes and with different MoAs. All eleven tested compounds had the ability to reduce the in vitro E2-induced production of Vtg in a concentration-dependent manner. The potency of the tested compounds differed by four orders of magnitude based on the concentrations for 50% inhibition (IC50). The observed order of potency was 2,3,7,8-tetrachlorodibenzo-p-dioxin>4-hydroxytamoxifen>3,3′,4,4′,5-pentachlorobiphenyl>benzo(k)fluoranthene>3,3′,4,4′-tetrachlorobiphenyl>β-naphthoflavone>ZM 189.154>indeno[1,2,3-cd]pyrene>benzo(b)fluoranthene>benzo(a)pyrene>benzo(a)anthracene. The CA and IA models were able to predict the combined effects of mixtures of ER antagonists with similar MoA. The mixtures of certain ER-antagonists with different and/or complex MoA caused deviations from both the CA and the IA model by causing higher anti-oestrogenic activity than predicted from the potency of the compounds alone. The rationale for these deviations warrants additional studies to assess the potential impacts on the health of organisms.

Intérêt de la chirurgie précoce dans l’endocardite infectieuse

Some environmental compounds are known to have anti-oestrogenic activity and their modes of action (MoA) are believed to include competitive inhibition of 17β-estradiol (E2) binding to the oestrogen receptor (ER) or interference with ER-dependent processes. The presence of multiple compounds having the same MoA may cause concern, as exposure to multiple compounds at concentrations below their threshold for effect can interact with cellular targets to cause effects in combination. The combined effect of mixtures can be assessed using prediction models such as concentration addition (CA) and independent action (IA). The objective of the present study was to determine if the CA and IA prediction models could accurately characterise the combined effects of mixtures of ER antagonists in rainbow trout (Oncorhynchus mykiss) hepatocytes using the ER-mediated production of the oestrogenic biomarker vitellogenin (Vtg) as a screening assay. Model anti-oestrogens (4-hydroxytamoxifen and ZM 189.154) and environmentally relevant compounds (PCBs and PAHs) were tested to ensure inclusion of compounds from different chemical classes and with different MoAs. All eleven tested compounds had the ability to reduce the in vitro E2-induced production of Vtg in a concentration-dependent manner. The potency of the tested compounds differed by four orders of magnitude based on the concentrations for 50% inhibition (IC50). The observed order of potency was 2,3,7,8-tetrachlorodibenzo-p-dioxin > 4-hydroxytamoxifen > 3,3′,4,4′,5-pentachlorobiphenyl > benzo(k)fluoranthene > 3,3′,4,4′-tetrachlorobiphenyl > β-naphthoflavone > ZM 189.154 > indeno[1,2,3-cd]pyrene > benzo(b)fluoranthene > benzo(a)pyrene > benzo(a)anthracene. The CA and IA models were able to predict the combined effects of mixtures of ER antagonists with similar MoA. The mixtures of certain ER-antagonists with different and/or complex MoA caused deviations from both the CA and the IA model by causing higher anti-oestrogenic activity than predicted from the potency of the compounds alone. The rationale for these deviations warrants additional studies to assess the potential impacts on the health of organisms.

Assessing combined toxicity of estrogen receptor agonists in a primary culture of rainbow trout ( Oncorhynchus mykiss) hepatocytes

The presence of highly complex mixtures of chemicals in the environment challenges our ability to assess single chemical effects and the interaction that occurs with cellular receptor targets and regulation of endocrine processes. In this study concentration addition (CA) and independent action (IA) prediction models were used to assess the combined toxicity of mixtures of environmental relevant estrogen receptor (ER) agonists (hormones and anthropogenic pollutants) in a primary culture of rainbow trout ( Oncorhynchus mykiss) hepatocytes using the ER-mediated production of vitellogenin (Vtg) as a biological marker (biomarker) for estrogenicity. Nine of the eleven tested chemicals induced the production of Vtg and the parameters from the fitted concentration–response curves were used to model four mixtures containing four (17β-estradiol, estrone, estriol and diethylstilbestrol), five (musk ketone, 4- tert-octylphenol, bisphenol A, o,p′-DDT and dibenzothiophene), seven (17β-estradiol, estrone, estriol, diethylstilbestrol, 4- tert-octylphenol, bisphenol A and o,p′-DDT) and nine compounds (17β-estradiol, estrone, estriol, diethylstilbestrol, musk ketone, 4- tert-octylphenol, bisphenol A, o,p′-DDT and dibenzothiophene). The CA and IA prediction model proved to be a good estimation for the combined effect of mixtures of ER agonists at low relative mixture concentration (e.g. relative to the maximum mixture concentrations used), but a deviation from the prediction models was observed when exposing hepatocytes to high relative mixture concentrations. The CA and IA prediction models’ ability to predict the combined estrogenic effect of complex mixtures, especially in the low concentration–response range, is of ecological relevance since organisms in the environment generally encounter low concentrations of chemicals from a wide array of chemical groups that may not elicit estrogenic effects on their own.

Evidence of feminization in wild Elliptio complanata mussels in the receiving waters downstream of a municipal effluent outfall

The endocrine-disrupting activity of municipal effluents has the potential to alter the reproductive system and induce feminization to aquatic organisms. The purpose of this study was to examine the sex ratio, vitellogenin (Vtg)-like proteins, serotonin, arachidonate cyclooxygenase (COX) activity and dopamine status in wild mussels living at sites upstream and downstream of two municipal effluent outfalls in the Mille-Îles River (Quebec, Canada). Gonad integrity was also studied by monitoring the gonado-somatic index (GSI), the activity of the rate-limiting enzyme aspartate transcarbamoylase (ATC) for purine synthesis, and changes in lipid peroxidation (LPO). The results showed that the proportion of females was dramatically increased from 30% at the upstream sites to 80% at the downstream sites. The levels of Vtg-like proteins were significantly elevated in the male mussels only. Male mussels downstream of the municipal effluent plumes expressed female-specific protein bands (Vtg-like), as determined by high-resolution gel electrophoresis and silver staining. The serotonin/dopamine ratio was significantly decreased in the downstream mussels, indicating that the gonad was in a state of early vitellogenesis. However, this change was not accompanied by changes in ATC, suggesting no significant egg production was underway; this was confirmed by the observation that the downstream mussels displayed significantly low GSIs. GSIs were rather dependent on the serotonin/dopamine ratio (r = 0.44; p < 0.001), while Vtg-like proteins were dependent on dopamine levels (r = 0.50; p < 0.001). The increase in COX activity at the downstream sites and its close relationship with increased serotonin levels suggest a concomitant serotonergic signalling in addition to VTG production. The production of Vtg-like proteins combined with the serotonergic effects of the municipal effluents was associated with oxidative damage (LPO) in the gonad. This study provides the first evidence of feminization in wild mussel populations and the disruption in gonad physiology by exposure to municipal effluents.

Effects of monocrotophos on the reproductive axis in the male goldfish ( Carassius auratus): Potential mechanisms underlying vitellogenin induction

Monocrotophos (MCP) is a highly toxic organophosphorus pesticide that has been banned in many countries. Both vitellogenin mRNA expression and secretion were significantly induced in male goldfish by exposure to an MCP-based pesticide, suggesting that MCP has significant estrogenic properties. To elucidate the mechanisms of action of MCP on vitellogenin induction, we used radioimmunoassay to examine the effect of MCP treatment on plasma 17β-estradiol and testosterone levels in male goldfish ( Carassius auratus). We also investigated the potential impacts of MCP treatment on aromatase expression, on the synthesis and secretion of pituitary gonadotropins and on the regulation of hypothalamic gonadotropin-releasing hormones by real-time PCR and radioimmunoassay. Experiments were carried out during the period of gonadal late recrudescence following a 21-day exposure to 0.01, 0.10 and 1.00 mg L −1 of a pesticide containing 40% MCP in a semi-static exposure system. The results indicated that males in each MCP treatment group had much higher plasma levels of 17β-estradiol, suggesting that the induction of VTG production by MCP was indirectly caused by elevated levels of endogenous 17β-estradiol. MCP-induced plasma 17β-estradiol levels via interference with the reproductive axis at multiple potential sites in male goldfish: (a) MCP exposure enhanced the mRNA expression of gonadal aromatase, the enzyme that converts androgens into estrogens, consequently reducing plasma levels of testosterone and increasing plasma concentrations of 17β-estradiol; (b) MCP treatment increased follicle-stimulating hormone β subunit mRNA expression and protein secretion and decreased luteinizing hormone β subunit mRNA expression and protein secretion, thus interfering with gonadotropin synthesis and secretion at the pituitary level and leading to the disruption of reproductive endocrine control and androgen and estrogen balance.

Interacting effects of tributyltin and 17β‐estradiol in male Chinese loach (Misgurnus anguillicaudatus)

In this study, male Chinese loaches in a semistatic waterborne exposure system were used to study the effects of tributyltin (TBT) on vitellogenin (Vtg) production induced by 17beta-estradiol (E2), TBT accumulation and distribution in tissues, and the effects of E2 on the distribution of TBT. The results demonstrate that TBT does not induce the synthesis of Vtg in male Chinese loach and that TBT could significantly inhibit Vtg production induced by E2, after exposure to binary mixtures of E2 and TBT for 14 days. TBT was found to accumulate in the liver, kidney, gills, intestines, and muscles of male Chinese loach, wherein the liver, kidney, gills, and intestines were found to have the most TBT accumulation. The existence of E2 did not significantly affect tissue distribution of TBT.

Effects of adrenergic agonists on the extrahepatic expression of vitellogenin Ao1 in heart and brain of the Chinese rare minnow ( Gobiocypris rarus)

Teleost vitellogenins (VTGs) are large multidomain apolipoproteins and traditionally considered as the estrogen responsive precursors of the major egg yolk proteins. We identified five clones encoding VTGs, about 16% of the random EST clones from our constructed cDNA library from Chinese rare minnow liver tissue treated with 17β-estradiol (E2). Full-length vtgAo1 has been obtained based on the sequence information of four partial cDNA inserts by RACE. The inducibility of the vtgAo1 expression in liver by E2 was confirmed by RT-PCR. The presence of vtgAo1 transcripts have been observed primarily in liver. However, a significant level of the vtgAo1 was found in an unexpected location, heart, particularly in atrial cells by RT-PCR and whole mount in situ hybridization analyses. The vtgAo1 mRNA expression in heart and liver tissue could be suppressed by both α-adrenergic agonist, phenylephrine (PE) and β-adrenergic agonist, isoproterenol (ISO). The expression of VTG in the heart observed in the present studies suggested it may provide protection from surplus intracellular lipids in fish cardiomyocytes as triglyceride transport proteins do in mammals. The results also indicated that the production of teleost vtg in vivo can be regulated by not only estrogenic agents, but adrenergic signals as well.