Vitellogenin Induction Research Articles

Effects of individual and binary mixtures of estrogens on male goldfish (Carassius auratus).

Adverse effects of five typical environmental estrogens, namely estrone (E1), 17β-estradiol (E2), 4-n-octylphenol (4-n-OP), 4-n-nonylphenol (4-n-NP) and bisphenol A (BPA) on adult male goldfish (Carassius auratus) were investigated both individually and in binary mixtures, using serum vitellogenin (VTG) induction and gonadosomatic index (GSI) as the endpoints. Doses of individual and binary mixtures of estrogens were chosen at broad ranges. Five individual estrogens induced common dose-dependent increases of serum VTG in the experimental fish when injection doses of the estrogen series were comparatively low. The levels of VTG induction in fish descended after peaked at a certain dose of the individual estrogen. Significant GSI decreases were observed in fish treated by all dose series of E1 and E2, and comparatively high doses of 4-n-OP, 4-n-NP and BPA when compared with that of solvent control (SC). Effects of binary mixtures of the five typical estrogens on VTG induction in male goldfish were in additive manner at low-effect doses, but divergences occurred at high dose levels, with the predicted effects by additive manner exceeding those were observed. All of GSI of fish treated by the binary mixtures were about or lower than 10(-3)%. Serious atrophy of gonads was observed in all the mixture treatment groups when compared with that of SC. These findings highlight the potential reproductive risk of fish resulted from existing mixtures of hormones in the aquatic environment, and they have important implications for environmental estrogen hazard assessment.

Effects of combined exposure to 17α-ethynylestradiol and dibutyl phthalate on the growth and reproduction of adult male zebrafish (Danio rerio)

To evaluate the combined effects of 17α-ethynylestradiol (EE2) and dibutyl phthalate (DBP) on the growth and reproduction of male zebrafish, three-month-old fish were exposed to 0.005 or 0.020µg/L EE2, 100 or 500µg/L DBP or their binary mixtures under semi-static conditions. Investigated parameters include the length, weight, condition factor, vitellogenin (VTG) induction, acyl-CoA oxidase (AOX) protein level, histopathological alteration of testis, liver and gill, and reproductive capacity. After 21d exposure, no statistical difference was found among the weights, lengths and condition factors of different treatment groups. In all binary mixture groups, decreased VTG levels were detected compared to EE2-only groups; and the AOX levels were significantly lower than DBP-only treatments while both chemicals can individually induce AOX synthesis. Therefore, EE2 and DBP may act additively on VTG and antagonistically on AOX induction in males. After 45d exposure, delayed gametogenesis was observed for the DBP-only groups, indicated by fewer spermatozoa and more spermatocytes, which was further aggravated with the addition of EE2. The developmental delay of testis partially recovered after a 30d depuration in clean water. Combined exposure also caused liver and gill lesions, which were not alleviated during the 30d depuration, suggesting a nonreversible harmful effect the same as single exposure. Mixed EE2 and DBP were observed to impair the reproductive capability (the fecundity and fertilization rate) of males, while single exposure did not. Co-exposed to 0.020µg/L EE2 and 100µg/L DBP promoted the early hatching of offspring (F1 generation) at 48h post-fertilization (hpf), but the survival rates of the F1 generation were similar in all treatments. Our findings indicate that the effects of mixed EE2 and DBP at environmentally relevant levels can be either antagonistic or additive relying on the specific toxicological endpoints and the respective doses of each chemical.

Vitellogenin of the northern leopard frog (Rana pipiens): Development of an ELISA assay and evaluation of induction after immersion in xenobiotic estrogens

An immunoassay for leopard frog (Rana pipiens) vitellogenin was developed for studying endocrine disruption. Male frogs were injected with estradiol-17β to stimulate vitellogenin for purification. SDS–PAGE revealed high amounts of a 170–180kDa protein, which was confirmed to be vitellogenin by Western blotting. Vitellogenin was purified by DEAE chromatography and used to generate a polyclonal antibody. A competitive ELISA was developed for leopard frog vitellogenin with a detection limit of 6.0ngmL−1 and a working range of 20–1000ngmL−1. The intra-assay coefficient of variation averaged 5.47% for control sera and 9.71% for estrogen-treated sera. The inter-assay coefficient of variation averaged 8.21% for control sera and 9.93% for estrogen-treated sera. Recovery of purified vitellogenin averaged 95.2%. Vitellogenin was measured in male frogs immersed in the estrogenic compound diethylstilbestrol (DES) for various times and doses. Serum vitellogenin was detected within five days after immersion in 1.0mgL−1 DES and levels continued to increase through 20d. In a 20-day dose–response experiment, serum vitellogenin was detected in frogs immersed in 0.01mgL−1 DES and vitellogenin concentration increased with dose. Immersion of frogs in one of several xenobiotic estrogens (nonylphenol, octylphenol, bisphenol-A) for 20d did not increase vitellogenin for any treatment, suggesting that this frog may be less sensitive than fish to endocrine disruptors. Vitellogenin induction in R.pipiens may be a useful amphibian model system for field studies of endocrine disruption, due to its broad geographic range.

Molecular responses to 17β-estradiol in early life stage salmonids

Environmental estrogens (EE) are ubiquitous in many aquatic environments and biological responses to EEs in early developmental stages of salmonids are poorly understood compared to juvenile and adult stages. Using 17β-estradiol (E2) as a model estrogen, waterborne exposures were conducted on early life stage rainbow trout (Oncorhynchus mykiss; egg, alevin, swim-up fry) and both molecular and physiological endpoints were measured to quantify the effects of E2. To investigate developmental stage-specific effects, laboratory exposures of 1μg/L E2 were initiated pre-hatching as eyed embryos or post-hatching upon entering the alevin stage. High mortality (∼90%) was observed when E2 exposures were initiated at the eyed embryo stage compared to the alevin stage (∼35% mortality), demonstrating stage-specific sensitivity. Gene expression analyses revealed that vitellogenin was detectable in the liver of swim-up fry, and was highly inducible by 1μg/L E2 (>200-fold higher levels compared to control animals). Experiments also confirmed the induction of vitellogenin protein levels in protein extracts isolated from head and tail regions of swim-up fry after E2 exposure. These findings suggest that induction of vitellogenin, a well-characterized biomarker for estrogenic exposure, can be informative measured at this early life stage. Several other genes of the reproductive endocrine axis (e.g. estrogen receptors and androgen receptors) exhibited decreased expression levels compared to control animals. In addition, chronic exposure to E2 during the eyed embryo and alevin stages resulted in suppressive effects on growth related genes (growth hormone receptors, insulin-like growth factor 1) as well as premature hatching, suggesting that the somatotropic axis is a key target for E2-mediated developmental and growth disruptions. Combining molecular biomarkers with morphological and physiological changes in early life stage salmonids holds considerable promise for further defining estrogen action during development, and for assessing the impacts of endocrine disrupting chemicals in vivo in teleosts.

Understanding Receptor-Mediated Effects in Rainbow Trout:In Vitro–in VivoExtrapolation Using Physiologically Based Toxicokinetic Models

The European REACH regulation requires the use of animal experimentation to assess the risk of industrial chemicals. However, the 3R principle (reduction, replacement, refinement) demands the use of suitable alternative test methods. Many dossiers submitted for the authorization of chemicals have attempted to provide the required data without performing new experiments, relying heavily on in silico methods; in vitro assays were scarcely used. We propose a methodology that uses physiologically based toxicokinetic (PBTK) models to extrapolate in vitro data to the in vivo level. We collected experimental results for in vitro and in vivo ethoxyresorufin-O-deethylase and vitellogenin induction following chemical exposure and compared those results with model predictions. We found that the predictive power of aqueous chemical concentrations was limited; median effect concentrations (EC50s) based on internal concentrations in fish correlated better with in vitro EC50s. Our data show that in vitro assays could offer a substitute for fish studies when combined with PBTK models.

Estrogenic effects of monocrotophos standard on male goldfish, Carassius auratus: induction of vitellogenin synthesis and alteration of testicular ultrastructure and function

Monocrotophos has been shown to exert estrogenic effects on teleosts. However, it has not been determined whether monocrotophos itself is the component that is responsible for exerting these estrogenic activities. Therefore, this study was carried out to investigate the estrogenic effects of monocrotophos standard in male goldfish (Carassius auratus), after a 21-day semi-static toxicity test. The induction of vitellogenin synthesis in males exposed to monocrotophos was confirmed with qualitative and quantitative methods. The observed increase in endogenous estrogen levels in males exposed to monocrotophos might explain the induction of vitellogenin. Moreover, we observed a decreased gonadosomatic index, ultrastructure damage of sperm and Sertoli cells, and several alterative testicular enzyme activities in males exposed to monocrotophos, which possibly affect spermatogenesis and male fertility. Our study showed the potential estrogenic effects of monocrotophos standard may act via the induction of vitellogenin synthesis and alteration of testicular ultrastructure and function in male goldfish.

Estrogenic chemical effects are independent from the degree of sex role reversal in pipefish

Endocrine disrupting chemicals (EDCs) have been reported to disturb several ecological relevant endpoints. Surprisingly, EDC-induced effects on fish sexual behaviour have been poorly studied despite the fact that even subtle alterations might contribute to a disruption of sexual interactions, thus negatively impacting reproduction. As the few assessments on sexual behaviour have been conducted in species with orthodox sex roles, it might be argued that sex-role reversed species might provide a potentially complementary system to further explore the effects of EDCs on reproduction. In the present study, two pipefish species with distinct degrees of sex-role reversal were selected to further elucidate the impact of chronic EE2 exposure on sexual behaviour and reproduction-related endpoints. The obtained results indicate that, independently of the degree of sex role reversal, courtship behaviour seems to resist oestrogenic chemical exposure. However, exposure to environmentally relevant EE2 levels did induce a complete absence of pregnancies at 18ng/L. Even though pregnancies were observed at intermediate concentrations, the percentage of non-transferred or misplaced oocytes increased and a dose-dependent decrease of oocyte volume was observed. Imbalances in the oogenesis process, induction of vitellogenin in males and the absence of pregnancies highlight that environmental relevant concentrations of EE2 have the potential to negatively affect pipefish populations, most of them inhabiting coastal areas where oestrogenic contamination is more prevalent.

Estrogenic effect of the phytoestrogen biochanin A in zebrafish, Danio rerio, and brown trout, Salmo trutta

Isoflavones with estrogenic activity produced in Fabaceae plants are known to leach from agricultural areas to freshwater systems, but the effect of waterborne isoflavones in fish has not been thoroughly characterized. Therefore, the estrogenic effect of waterborne biochanin A was investigated in zebrafish (Danio rerio) and juvenile brown trout (Salmo trutta). Exposure of juvenile brown trout to 10μgbiochanin AL−1 or higher caused marked vitellogenin induction after 9–10 days of exposure and so did exposure to 186μgbiochanin AL−1 for 6h. Following 8d of exposure, a NOEC for induction of vitellogenin production in male zebrafish was 70 and LOEC 114μgbiochanin AL−1. Exposure to 209μgbiochanin AL−1 from hatch to 60 days post hatch (dph) caused a skewing of the sex ratio toward more phenotypic female zebrafish, but did not cause induction of vitellogenin in male and undifferentiated fish. In conclusion: (1) biochanin A elicits estrogenic effects in trout at environmentally realistic concentrations, (2) brown trout plasma vitellogenin concentrations respond to lower biochanin A exposure concentrations than vitellogenin concentrations in zebrafish homogenates and (3) concerning vitellogenin induction, the hypothesis should be tested if short term tests with zebrafish may show a higher sensitivity than partial life cycle tests.

Environmental estrogens in an urban aquatic ecosystem: II. Biological effects

Urban aquatic ecosystems are often overlooked in toxicological studies even though they serve many ecosystem functions and sustain fish populations despite large-scale habitat alterations. However, urban fish populations are likely exposed to a broad range of stressors, including environmental estrogens (EEs) that may affect anatomy, physiology and reproduction of exposed fish. Although significant progress has been made in establishing ecological consequences of EE exposure, these studies have focused largely on hydrologically simple systems that lack the complexity of urban aquatic environments. Therefore, the objective of this study was to assess the occurrence and biological effects of EEs across a large urbanized aquatic ecosystem. A multi-pronged study design was employed relying on quantitative determination of select EEs by liquid chromatography tandem mass spectrometry and repeated biological monitoring of wild-caught and caged fish for indications of endocrine disruption. Over three years, EEs were measured in aqueous samples (n=42 samples) and biological effects assessed in >1200 male fish across the 2000km2 aquatic ecosystems of the Greater Metropolitan Area of Chicago, IL. Our study demonstrated that in addition to water reclamation plant (WRP) effluents, non-WRP sources contribute significant EE loads to the aquatic ecosystem. While resident and caged male fish responded with the induction of the egg-yolk protein vitellogenin, an indicator of EE exposure, neither resident nor caged sunfish exhibited prevalent histopathological changes to their reproductive organs (i.e., intersex) that have been reported in other studies. Vitellogenin induction was greater in spring than the fall and was not correlated with body condition factor, gonadosomatic index or hepatosomatic index. Exposure effects were not correlated with sites downstream of treated effluent discharge further affirming the complexity of sources and effects of EEs in urban aquatic ecosystems.

Single and Combined Effects of Estrone and 17β-Estradiol on Male Goldfish

ObjectiveTo assess the single and combined effects of estrone (E1) and 17β-estradiol (E2) on goldfish (Carassius auratus). MethodsBatch tests were conducted. Serum levels of vitellogenin (VTG) and E2, gonadosomatic indices (GSI), gonadal DNA damage and liver 7-ethoxyresorufin-O-deethylase (EROD) activity were measured after exposure for 14 days. ResultsThe VTG level increased significantly in a concentration-dependent manner. The serum E2 level was significantly higher and the GSI level was significantly lower in goldfish after exposed to the 3 drugs. DNA damage occurred in treated samples and EROD activity was significantly suppressed 7 days after exposure. The joint effect of E1 and E2 was additive with regard to VTG induction. ConclusionThe results of our study highlight a series of effects of steroidal estrogens on goldfish. Further study is needed to confirm their effect as a whole.

Morpholino-Mediated Knockdown of ERα, ERβa, and ERβb mRNAs in Zebrafish (Danio rerio) Embryos Reveals Differential Regulation of Estrogen-Inducible Genes

Genetically distinct estrogen receptor (ER) subtypes (ERα and ERβ) play a major role in mediating estrogen actions in vertebrates, but their unique and overlapping functions are not entirely clear. Although mammals have 1 gene of each subtype (ESR1 and ESR2), teleost fish have a single esr1 (ERα) and 2 esr2 (ERβa and ERβb) genes. To determine the in vivo role of different ER isoforms in regulating estrogen-inducible transcription targets, zebrafish (Danio rerio) embryos were microinjected with esr-specific morpholino (MO) oligonucleotides to disrupt splicing of the exon III/intron III junction in the DNA-binding domain. Each MO knocked down its respective normal transcript and increased production of variants with a retained intron III (esr1 MO) or a deleted or mis-spliced exon III (esr2a and esr2b MOs). Both esr1 and esr2b MOs blocked estradiol induction of vitellogenin and ERα mRNAs, predominant hepatic genes, but esr2b was the only MO that blocked induction of cytochrome P450 aromatase B mRNA, a predominant brain gene. Knockdown of ERβa with the esr2a MO had no effect on estrogen induction of the 3 mRNAs but, when coinjected with esr1 MO, attenuated the effect of ERα knockdown. Results indicate that ERα and ERβb, acting separately or cooperatively on specific gene targets, are positive transcriptional regulators of estrogen action, but the role of ERβa, if any, is unclear. We conclude that MO technology in zebrafish embryos is an advantageous approach for investigating the interplay of ER subtypes in a true physiological context.

Assessment of In Vivo Estrogenic Response and Identification of Environmental Estrogens in Influent and Effluent from a Sewage Treatment Plant

The in vivo estrogenic response and estrogenic contents of the influent and effluent collected from a sewage treatment plant located in Jiaozuo were assessed. The bioassay showed significant serum vitellogenin (VTG) induction in all the treated male goldfish (Carassius auratus) and significant gonad atrophies were only observed in the fish induced the most VTG expressions. Six target estrogens (estrone, 17β-estradiol, 17α-ethynylestradiol, 4-n-octylphenol, 4-n-nonylphenol and bisphenol A) were detected in different polar fractions, with the exception of the 25 % and 50 % methanol fractions extracted from the influent and the 25 %, 50 %, 95 % and 100 % methanol fractions extracted from the effluent. For both the influent and effluent, natural and synthetic steroidal estrogens were detected in those extracted fractions induced the most abundant VTG expressions.

Molecular Cloning, Characterization, and Expression Pattern of the Ultraspiracle Gene Homolog (RXR/USP) from the Hemimetabolous Insect Periplaneta americana (Dictyoptera, Blattidae) During Vitellogenesis

Ecdysteroid and sequiterpenoids juvenile hormones play a gonadotrophic role in the insect adult female vitellogenesis. The molecular basis of hormone action has been analyzed in great detail in flies and moths, but rarely in primitive insect orders. The primitive hemimetabolous insect Periplaneta americana was used, as a model, to isolate and characterize, for the first time, two cDNAs of RXR/USP, a component of the heterodimeric ecdysone receptor. These two cDNAs correspond to two isoforms, named PamRXR-S (short form) and PamRXR-L (long form). Both are identical except for 25 amino acids deletion/insertion located in the loop between helices H1 and H3 of the ligand-binding domain. The two isoforms are differentially expressed in different tissues as revealed by RT-PCR and northern blot analysis. In fat body, brain, ovary, and muscle tissues, the predominant form was PamRXR-S, whereas PamRXR-L was abundant in ovaries. The PamRXR transcript was detected during all stages of vitellogenesis in the fat body with different levels. It was little low during the early vitellogenic period (days 2, 3), then a peak of increase was detected during days 4-6 (day 5) which was followed by another peak of increase at the end of vitellogenesis, day 9. We assumed that PamRXR might play a dual role of induction of vitellogenin through JH at early vitellogenesis and suppression through 20E during late vitellogenesis. The present work will pave the way for several other investigations to understand both the ecdysteroid-dependent genetic hierarchy and JH mechanism controlling vitellogenesis in the American cockroach, P. americana.

Transgenic (cyp19a1b-GFP) zebrafish embryos as a tool for assessing combined effects of oestrogenic chemicals

Endocrine disrupting chemicals and especially oestrogen receptor (ER) agonists have been extensively studied over the years due to their potential effects on sexual development and reproduction in vertebrates, notably fish. As ER agonists can exist as complex mixtures in the aquatic environment, evaluating the impact of combined exposure on oestrogenic effects has become increasingly important. Use of predictive models such as concentration addition (CA) and independent action (IA) has allowed assessment of combined estrogenic effects of complex multi-compound mixtures of ER agonists in various fish in vitro and in vivo experimental models. The present work makes use of a transgenic zebrafish strain, tg(cyp19a1b-GFP), which expresses the green fluorescent protein (GFP) under the control of the cyp19a1b (brain aromatase or aromatase B) gene to determine the oestrogenic potency of ER agonists alone or in mixtures. In these studies, tg(cyp19a1b-GFP) zebrafish embryos were exposed for four days (from one to five days post fertilization) to five different oestrogenic chemicals; 17α-ethinylestradiol (EE2), 17β-estradiol (E2), estrone (E1), bisphenol A (BPA) and 4-tert-octylphenol (OP), and three mixtures of up to four of these compounds. The mixture of BPA, OP and E2 was also tested with primary cultures of rainbow trout hepatocytes by analysing the ER-mediated induction of the oestrogenic biomarker vitellogenin in order to compare the performance of the two methods for assessing oestrogenic effects of complex mixtures. The three tested mixtures were predominantly acting in an additive manner on the expression of GFP. Additivity was indicated by the overlap of the 95% confidence interval of the concentration response curves for the observed data with the CA and IA prediction models, and model deviation ratios within a factor of two for a majority of the mixture concentrations. However, minor deviations determined as more than additive effects for the mixture of EE2, E1 and E2 and less than additive effects for the mixture of BPA, OP, EE2 and E1 were observed at the higher mixture concentrations tested. The successful prediction of additivity by CA and IA in tg(cyp19a1b-GFP) zebrafish embryos and deviations at high mixture concentrations seemed to correspond well to results obtained in the rainbow trout hepatocyte assay. The present results clearly show the usefulness of combining predictive modelling and use of in vitro bioassays for rapid screening of oestrogenic effects of complex mixtures and environmental samples.

Induction of vitellogenin synthesis in immature male yellowfin seabream (Acanthopagrus latus) exposed to 4-nonylphenol and 17β-estradiol

In the present study, the effects of xenoestrogen 4-nonylphenol (4-NP) and 17β-estradiol (E2) on the synthesis of vitellogenin (VTG) in immature male yellowfin seabream (Acanthoparus latus) were assessed to determine the potential use of this species as a biological model for studying endocrine disrupters in the Musa estuary in southern seawaters of Iran. A total number of 104 fish were injected by 10, 50, 100, 150 and 200 µg g(-) (1) week(-) (1) of 4-NP and 2 µg g(-) (1) week(-) (1) of E2, and their blood plasma was obtained for biochemical analysis on days 0, 7 and 14. The hepatosomatic index (HSI) of each fish was also recorded at each sampling time. The results showed that 4-NP significantly induced VTG synthesis in A. latus, which was detected by gel electrophoresis as two major protein bands (∼210 and 191 KDa). The induction of VTG was also determined indirectly by measuring the alkali-labile phosphate, total plasma calcium and protein. All of these VTG indicators significantly increased in the plasma of 4-NP- and E2-treated fish. Meanwhile, a significant dose-dependent increase was observed in HSI, indicating the activation of hepatic VTG production in 4-NP- and E2-injected fish. Furthermore, the plasma alanine aminotransferase and aspartate aminotransferase concentrations significantly increased in 4-NP-treated fish, presumably reflecting a situation of hepatic tissue damage due to 4-NP toxicity. Based on the results, it is suggested that A. latus could be used successfully as a model species for future studies of endocrine disrupting contaminants in the Musa estuary.

The maturity index as a tool to facilitate the interpretation of changes in vitellogenin production and sex ratio in the Fish Sexual Development Test

In July 2011, the Fish Sexual Development Test (FSDT) has officially been adopted as OECD test guideline 234 for the detection of endocrine disrupting chemicals (EDCs). Sex ratio and vitellogenin (VTG) induction are the mandatory endocrine endpoints within this test, whereas gonad staging is only included as an option. In the present study, five FSDTs with zebrafish (Danio rerio) were conducted with EDCs with different modes of action (17α-ethinylestradiol, dihydrotestosterone, 17β-trenbolone, prochloraz and 4-tert-pentylphenol). Results document that not only sex ratio and VTG production of the exposed fish were massively affected, but also gonad maturation. As a novel approach for the quantification of gonad maturation in zebrafish, the maturity index was developed to allow not only an improved assessment of dose-dependent EDC-related effects on gonad maturation, but also statistical analysis of histological data. VTG induction and maturity index showed an excellent correlation for all five EDCs tested. Most importantly, the maturity index often helped to find appropriate interpretations for results that seemed contradictory at first sight. Results show that histological analyses and their predictive power for population fitness are currently underestimated and should become a standard component in the evaluation of potential EDCs.

Annual and seasonal evaluation of reproductive status in hornyhead turbot at municipal wastewater outfalls in the Southern California Bight

Treated wastewater effluent containing endocrine-disrupting chemicals is discharged into the coastal waters of the Southern California Bight (SCB) daily. The present study investigated changes in indicators of reproductive health and environmental estrogen exposure in hornyhead turbot (Pleuronichthys verticalis) near wastewater outfalls. Fish were collected from discharge areas, farfield stations, and a reference location in the SCB to examine spatial and temporal patterns. Fish from the Orange County outfall farfield site were younger and less sexually mature than fish from other sites. The sex ratio was skewed in some fish from outfall sites as well as from the Dana Point reference site. However, no consistent pattern in sex ratio was present over time. Low-level induction of vitellogenin was frequently observed in male fish from all sites, suggesting widespread exposure to estrogenic compounds, but did not appear to impact reproductive function as there was no incidence of gonad abnormalities (ova-testis). Analysis of historical hornyhead turbot trawl data indicated that populations are either increasing or stable in the SCB; thus, environmental estrogen exposure was not adversely impacting fish abundance. Additional research is needed to determine the cause of the estrogenic response in hornyhead turbot and whether the source of the estrogenic compounds is a consequence of historical contamination or of ongoing sources or representative of baseline characteristic of this species.

Laccase-Mediated Transformations of Endocrine Disrupting Chemicals Abolish Binding Affinities to Estrogen Receptors and Their Estrogenic Activity in Zebrafish

Endocrine disrupting chemicals (EDCs) are known to mainly affect aquatic organisms, producing negative effects in aquaculture. Transformation of the estrogenic compounds 17β-estradiol (E2), bisphenol-A (BPA), nonylphenol (NP), and triclosan (TCS) by laccase of Coriolopsis gallica was studied. Laccase is able to efficiently transform them into polymers. The estrogenic activity of the EDCs and their laccase transformation products was evaluated in vitro as their affinity for the human estrogen receptor alpha (hERα) and for the ligand binding domain of zebrafish (Danio rerio) estrogen receptor alpha (zfERαLBD). E2, BPA, NP, and TCS showed higher affinity for the zfERαLBD than for hERα. After laccase treatment, no affinity was found, except a marginal affinity of E2 products for the zfERαLBD. Endocrine disruption studies in vivo on zebrafish were performed using the induction of vitellogenin 1 as a biomarker (VTG1 mRNA levels). The use of enzymatic bioreactors, containing immobilized laccase, efficiently eliminates the endocrine activity of BPA and TCS, and significantly reduces the effects of E2. The potential use of enzymatic reactors to eliminate the endocrine activity of EDCs in supply water for aquaculture is discussed.

Pathogenic infection confounds induction of the estrogenic biomarker vitellogenin in rainbow trout

To examine the behavior of the estrogenic biomarker vitellogenin (VTG) under the combined impact of estrogens and pathogens, parasite-infected or noninfected rainbow trout were exposed to two doses of 17β-estradiol (E2). Infected and E2-exposed fish showed significantly lower hepatic VTG mRNA levels than healthy fish. Transcriptome data suggest that this was due to energetic constraints. Reduced responsiveness of the VTG biomarker in parasitized fish might obscure detection of low-level field exposure.

Toxicity of atrazine and nonylphenol in juvenile rainbow trout (Oncorhynchus mykiss): Effects on general health, disease susceptibility and gene expression

Atrazine (ATZ) and nonylphenol (NP) are commonly identified contaminants in aquatic habitats; however, few studies have considered the impact of these endocrine disrupters on immune function and resistance to disease. This study examined the immunotoxicological effects of ATZ and NP at multiple levels of biological organization. Juvenile rainbow trout (Oncorhynchus mykiss) were exposed to a solvent control (0.00625%, v/v anhydrous ethanol), or sub-lethal concentrations of ATZ (59μg/L and 555μg/L) or NP (2.3μg/L or 18μg/L) for 4d. At the end of exposure, fish were assessed for a number of physiological endpoints, including a host resistance challenge, and liver gene expression was assessed using a salmonid microarray (cGRASP, 32K version 1). While the low ATZ and low NP treatments had no measurable effects on the physiological endpoints measured, fish exposed to the high ATZ concentration (555μg/L) exhibited significantly elevated plasma cortisol, a decrease in SSI, and decreased lymphocytes and increased monocytes in peripheral blood, with suppression of early immune system processes apparent at the molecular level. In contrast, fish exposed to the high NP concentration (18μg/L) showed physiological (e.g. significantly elevated LSI) and gene expression changes (e.g. induction of vitellogenin) consistent with estrogenic effects, as well as decreased lymphocytes in the peripheral blood and more limited alterations in immune system related pathways in the liver transcriptome. Fish exposed to high ATZ or NP concentrations incurred higher mortality than control fish following a disease challenge with Listonella anguillarum, while fish exposed to the lower concentrations were unaffected. Microarray analysis of the liver transcriptome revealed a total of 211 unique, annotated differentially regulated genes (DRGs) following high ATZ exposure and 299 DRGs following high NP exposure. Functional (enrichment) analysis revealed effects on immune system function, metabolism, oxygen homeostasis, cell cycle, DNA damage, and other processes affected by ATZ or NP exposure. Overall, this study provides evidence at multiple levels of biological organization that both ATZ and NP are immunotoxic at sub-lethal concentrations and highlights the potential risk posed by these chemicals to wild fish populations.