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• ATBC exposure did not induce intersex or sex change in Japanese medaka. • The vtg1 and vtg2 expression in the liver of XX was reduced after ATBC exposure. • ATBC is not an endocrine disrupting chemical with strong sex hormone activity. Plasticizers are broadly classified as phthalate or nonphthalate. Recently, acetyl tributyl citrate (ATBC), an environmentally friendly nonphthalate plasticizer, was revealed to have the ability to disrupt thyroid hormone activity in fish species. Therefore, we aimed to assess whether ATBC exhibits any sex hormone (i.e., androgenic or estrogenic) activities. First, we examined the effects of ATBC on gonadal sex differentiation. Subsequently, we analyzed the different expression of biomarker genes that respond to endocrine-disrupting chemicals (EDCs) with sexual hormone activity in the liver. We observed normal testes and ovaries after both XX and XY medakas were exposed to ATBC, indicating that ATBC is not an EDCs with strong sex hormone activity and that it does not induce intersex (testis-to-ova or ovo-to-testis) or sex changes in Japanese medaka. The vitellogenin 1 (vtg1) and vitellogenin 2 (vtg2) mRNA expression levels in the liver of XX medakas were significantly reduced compared with those in the control group, whereas the expression levels of these genes in the liver of XY medakas remained unchanged. Finally, we examined the changes in the expression of biomarker genes that respond to EDCs with sex hormone activity in the gonads. The expression levels of biomarker genes did not differ significantly from that of the control group, although the expression levels of gsdf mRNA tended to increase while that of aromatase mRNA tended to decrease in the ovary of XX medakas following ATBC exposure. Conversely, the expression levels of gsdf and aromatase mRNAs in the testis of XY medakas remained unchanged. These results suggest that ATBC does not exhibit estrogenic activity, although it may have weak androgenic activity or no sexual hormone activity. [ABSTRACT FROM AUTHOR]

Antifungal azoles are widely used in medicine, agriculture, and material protection and several antifungal azoles have been found in environmental samples. Although these compounds were designed to inhibit fungal enzymes such as lanosterol-14-demethylase (cytochrome P450 (CYP) 51), it is well established that the inhibitory actions of azoles are not specific for fungal CYP isozymes. We refined a gill filament assay to determine the inhibition of CYP1, measured as reduced 7-ethoxyresorufin- O -deethylase (EROD) activity, in rainbow trout ( Oncorhynchus mykiss ) gill tissue ex vivo . The advantage of this method is that both induction and inhibition of EROD are performed ex vivo . Among thirteen azoles studied, the five that caused the strongest inhibition of gill EROD activity at a concentration of 5 μM were selected for concentration–response assessment. These compounds (bifonazole, clotrimazole, imazalil, miconazole, and prochloraz) showed IC 50 values ranging from 0.1 to 1.5 μM. CYP19 (aromatase) inhibition was measured using microsomes from rainbow trout brains. Concentration-response curves for CYP19 inhibition were determined for letrozole, bifonazole, clotrimazole, imazalil, miconazole and prochloraz, which gave IC 50 values ranging from 0.02 to 3.3 μM. It was further found that mixtures of the five most potent azoles reduced both CYP1 and 19 catalytic activity in an additive fashion (IC 50  = 0.7 μM and 0.6 μM, in the respective assay). Bifonazole (IC 50  = 0.1 μM) is not previously known to inhibit CYP1 activity. The additive inhibition of CYP1 and CYP19 catalytic activity is an important finding of the present study. We conclude that this additive action of azoles could mediate adverse impacts on CYP regulated physiological functions in environmentally exposed fish. [ABSTRACT FROM AUTHOR]

The present study was conducted to determine the effects and mechanism of waterborne copper (Cu) exposure influencing ovary development and related hormones secretion in yellow catfish Pelteobagrus fulvidraco . To this end, two experiments were conducted. In Exp. 1, the partial cDNA sequences of three steroidogenesis-related genes (androgen receptor ( ar ), steroidogenic factor 1 ( sf-1 ) and steroidogenic acute regulatory protein ( star )) were firstly characterized from P. fulvidraco . The predicted amino acid sequences for the P. fulvidraco ar , sf-1 and star contained the main structural features characteristic in other species. In Exp. 2, P. fulvidraco were exposed to three waterborne Cu concentrations (control, 30 μg/l and 60 μg/l, respectively) for 56 days. Sampling occurred on day 28 and day 56, respectively. On day 28, the levels of serum sex-steroid hormones (FSH and LH) and the mRNA levels of steroidogenesis-related genes ( 3β-hsd , cyp11a1 , cyp17 , cyp19a , sf-1 and star ) were significantly increased in ovary of P. fulvidraco exposed to 30 μg Cu/l. The immunohistochemical analysis showed the positive reaction of ER, VTG and aromatase in low dose exposure group. These indicated that in low dose and relative short-term exposure, Cu was beneficial. In contrast, 60 μg Cu/l exposure significantly reduced the levels of serum FSH, LH, E2 and P, and the mRNA levels of ovarian 20β-hsd , cyp19a and er α in P. fulvidraco . On day 56, waterborne Cu concentration exposure reduced the levels of serum gonadotropins and sex hormones, and down-regulated the mRNA levels of steroidogenesis-related genes, indicating long-term Cu exposure had toxic effect on the secretion of sex-steroid hormone in P. fulvidraco . For the first time, our study cloned cDNA sequences of ar , sf-1 and star in P. fulvidraco, and demonstrated the effects and mechanism of waterborne Cu exposure influencing hormones secretion and synthesis in dose- and time-dependent manner in P. fulvidraco , which will help to understand the Cu-induced reproductive toxicity at both protein and transcriptional levels in fish. [ABSTRACT FROM AUTHOR]

The Fish Sexual Development Test (FSDT) is a non-reproductive test to assess adverse effects of endocrine disrupting chemicals. With the present study it was intended to evaluate whether gene expression endpoints would serve as predictive markers of endocrine disruption in a FSDT. For proof-of-concept, a FSDT according to the OECD TG 234 was conducted with the non-steroidal aromatase inhibitor fadrozole (test concentrations: 10 μg/L, 32 μg/L, 100 μg/L) using zebrafish ( Danio rerio ). Gene expression analyses using quantitative RT-PCR were included at 48 h, 96 h, 28 days and 63 days post fertilization (hpf, dpf). The selection of genes aimed at finding molecular endpoints which could be directly linked to the adverse apical effects of aromatase inhibition. The most prominent effects of fadrozole exposure on the sexual development of zebrafish were a complete sex ratio shift towards males and an acceleration of gonad maturation already at low fadrozole concentrations (10 μg/L). Due to the specific inhibition of the aromatase enzyme (Cyp19) by fadrozole and thus, the conversion of C19-androgens to C18-estrogens, the steroid hormone balance controlling the sex ratio of zebrafish was altered. The resulting key event is the regulation of directly estrogen-responsive genes. Subsequently, gene expression of vitellogenin 1 ( vtg1 ) and of the aromatase cyp19a1b isoform ( cyp19a1b ), were down-regulated upon fadrozole treatment compared to controls. For example, mRNA levels of vtg1 were down-regulated compared to the controls as early as 48 hpf and 96 hpf. Further regulated genes cumulated in pathways suggested to be controlled by endocrine mechanisms, like the steroid and terpenoid synthesis pathway (e.g. mevalonate (diphospho) decarboxylase ( mvd ), lanosterol synthase (2,3 -oxidosqualene-lanosterol cyclase ; lss ), methylsterol monooxygenase 1 ( sc4mol) ) and in lipid transport/metabolic processes ( steroidogenic acute regulatory protein ( star ), apolipoprotein Eb ( apoEb )). Taken together, this study demonstrated that the existing Adverse Outcome Pathway (AOP) for aromatase inhibition in fish can be translated to the life-stage of sexual differentiation. We were further able to identify MoA-specific marker gene expression which can be instrumental in defining new measurable key events (KE) of existing or new AOPs related to endocrine disruption. [ABSTRACT FROM AUTHOR]

Although bioaccumulation of tributyltin (TBT) in fish has been confirmed, information on possible effects of TBT on reproductive system of fish is still relatively scarce, particularly at environmentally relevant levels. To evaluate the adverse effects and intrinsic toxicological properties of TBT in male fish, we studied aromatase gene expression in the brain, sex steroid contents, primary and secondary sexual characteristics, and reproductive behavior in male guppies ( Poecilia reticulata ) exposed to tributyltin chloride at the nominal concentrations of 5, 50, and 500 ng/L for 28 days in a semi-static exposure system. Radioimmunoassay demonstrated that treatment with 50 ng/L TBT caused an increase in systemic levels of testosterone of male guppies. Gonopodial index, which showed a positive correlation with testosterone levels, was elevated in the 5 ng/L and 50 ng/L TBT treated groups. Real-time PCR revealed that TBT exposure had inhibiting effects on expression of two isoforms of guppy aromatase in the brain, and these changes at the molecular levels were associated with a disturbance of reproductive behavior of the individuals, as measured by decreases in frequencies of posturing, sigmoid display, and chase activities when males were paired with females. This study provides the first evidence that TBT can cause abnormalities of secondary sexual characteristics in teleosts and that suppression of reproductive behavior in teleosts by TBT is due to its endocrine–disrupting action as an aromatase inhibitor targeting the nervous system. [ABSTRACT FROM AUTHOR]

The aim of this study was to investigate the effects of progestins on the sex differentiation of zebrafish by measuring the sex ratio and transcriptions of genes related to sex differentiation ( Amh , Dmrt1 , Figa , Sox9a and Sox9b genes) as well as sex hormone levels and transcriptional expression profiles along the hypothalamic–pituitary–gonadal (HPG) and hypothalamic–pituitary–adrenal (HPA) axes in juvenile zebrafish. Exposure of zebrafish to 4, 33, 63 ng L −1 progesterone (P4) or 4, 34, 77 ng L −1 norgestrel (NGT) started at 20 days post fertilization (dpf) and ended at 60 dpf. The results showed that exposure to P4 caused a significant increase in proportion of females as well as significant down-regulation of Amh gene and up-regulation of Figa at a concentration of 63 ng L −1 . However, the shift in the sex ratio toward males was observed following exposure to 34 and 77 ng L −1 NGT, which came along with the significant induction of Dmrt1 gene and inhibition of Figa gene. The sex hormones in exposed fish were measured with estrone being detected only in the fish exposed to the highest P4 concentration; whereas estradiol and androstenedione were detected only in the fish of the control and lowest NGT concentration. Furthermore, the increase in females was associated with the significant up-regulation of several key genes controlling the synthesis of sex hormones (i.e., Cyp17 , Cyp19a1a and Hsd3b ) following exposure to 63 ng L −1 P4 whereas the significant down-regulation of Cyp11a1 , Cyp17 , Cyp19a1a and Hsd3b genes was observed in the male-biased populations caused by 34 and 77 ng L −1 NGT. The overall results imply that both P4 and NGT could significantly affect sex differentiation in zebrafish, and that changes may be reflected by altered sex hormone levels and transcriptional expression profiles of genes related to synthesis of sex hormones. [ABSTRACT FROM AUTHOR]

As a weak estrogenic chemical, bisphenol A (BPA) has been extensively studied for reproductive toxicity and the effects on the steroidogenesis. In the present study, we aim to explore the effects of BPA on epigenetic modification in rare minnow Gobiocypris rarus . We have detected the global and cyp19a1a gene specific DNA methylation in gonads of adult G. rarus under BPA exposure. The global DNA methylation level was significantly increased in testis of the male fish exposed to BPA for 7 days, and it was significantly increased in the ovary following 35-days exposure. DNA methyltransferases (DNMTs) catalyze the transfer of a methyl moiety from S-adenosyl- l -methionine to the cytosine of a CpG dinucleotide. The alteration of the detected dnmts mRNA expression could affect the global DNA methylation levels following 15 μg/L BPA exposure. Cytochrome P450 aromatase (CYP19A1A), is responsible for the conversion of androgens into estrogens, which plays a vital role in estrogen synthesis in gonads. In the present study, the methylation level of ovarian cyp19a1a gene was significantly suppressed and stimulated by 7- and 35-day BPA exposure, respectively. There was a significant negative correlation between cyp19a1a mRNA expression and methylation levels of the four CpGs at the 5′ flanking region in the ovary of adult G. rarus following BPA exposure. So we hypothesize that there are some association between the reproductive toxicity of BPA and the global DNA methylation under BPA exposure. And the alteration of cyp19a1a expression in female G. rarus by BPA might attribute to the change of its DNA methylation status. [ABSTRACT FROM AUTHOR]

The fungicide vinclozolin (VZ) is in use globally and known to disrupt reproductive function in male. The present study tested the hypothesis that VZ disrupts testicular function in goldfish (Carassius auratus) by affecting brain-pituitary-testis axis. Goldfish were exposed to 100, 400 and 800 µg/L VZ and 5 µg/L 17β-estradiol (E2) for comparison. In VZ treated goldfish, 11-ketotesteosterone (11-KT) secretion was changed depending on dose and duration period of treatment. Following 7 days of exposure, 11-KT was decreased in goldfish exposed to 800 µg/L VZ, while it was increased in goldfish exposed to 100 µg/L VZ after 30 days of exposure. Circulating E2 level was unchanged in VZ treated goldfish, however the E2/11-KT ratio was increased in a concentration-related manner. In E2 treated goldfish, circulatory 11-KT and E2 levels were decreased and increased, respectively, which resulted in an increase in the E2/11-KT ratio. Exposure to VZ at 100 µg/L caused a significant increase in the circulatory luteinizing hormone (LH) after 30 days. In E2 treated fish circulatory LH was decreased, significantly. Transcripts of genes encoding gonadotropin-releasing hormone and androgen receptor in the brain, and those of genes encoding LH and follicle-stimulating hormone receptors, StAR, CYP17, and 3β-HSD in the testis changed in VZ-treated goldfish depending on concentration and period of treatment. mRNA of genes encoding vitellogenin and estrogen receptor in the liver and cytochrome P450 aromatase in the brain were increased in E2-treated goldfish. The results suggest that VZ-induced changes in 11-KT were due to disruption in brain-pituitary-testis axis and provide integrated characterization of VZ-related reproductive disorders in male fish. [ABSTRACT FROM AUTHOR]

Xenoestrogens may persist in the environment by binding to sediments or suspended particulate matter serving as long-term reservoir and source of exposure, particularly for organisms living in or in contact with sediments. In this study, we present for the first time an effect-directed analysis (EDA) for identifying estrogenic compounds in a sediment sample using embryos of a transgenic reporter fish strain. In the tg(cyp19a1b-GFP) transgenic zebrafish strain, the expression of GFP (green fluorescent protein) in the brain is driven by an oestrogen responsive element in the promoter of the cyp19a1b (aromatase) gene. The selected sediment sample of the Czech river Bilina had already been analysed in a previous EDA using the yeast oestrogen screening assay and had revealed fractions containing estrogenic compounds. When normal phase HPLC (high performance liquid chromatography) fractionation was used for the separation of the sediment sample, the biotest with transgenic fish embryos revealed two estrogenic fractions. Chemical analysis of candidate compounds in these sediment fractions suggested alkylphenols and estrone as candidate compounds responsible for the observed estrogenic effect. Alkylphenol concentrations could partially explain the estrogenicity of the fractions. However, xenoestrogens below the analytical detection limit or non-targeted estrogenic compounds have probably also contributed to the sample's estrogenic potency. The results indicated the suitability of the tg(cyp19a1b-GFP) fish embryo for an integrated chemical-biological analysis of estrogenic effects. [ABSTRACT FROM AUTHOR]

Highlights: [•] Reduction in the sizes of the spermatocytes and spermatids in fish after treatment with di-n-butyl phthalate (DnBP). [•] Increase in the proportion of spermatogonia after exposure to DnBP. [•] Vitellogenin protein induction in 500μg/L and 1000μg/L treatment groups. [•] An induction in the genes coding for ERα, ERβ, ARα and ARβ after treatment with 1000μ/L DnBP. [ABSTRACT FROM AUTHOR]

Highlights: [•] Effects of four EDCs on reproduction and aromatase in a marine fish were studied. [•] Aromatase activity in brain and ovary were affected differently by the four EDCs. [•] Changes in male brain aromatase activity were associated with decreased reproduction. [•] GSI, but not plasma vitellogenin, mirrored changes in male brain aromatase activity. [Copyright &y& Elsevier]

Abstract: Fish embryos were used to evaluate the interaction among common environmental and chemical stressors found in urban coastal environments, namely hypoxia, aryl hydrocarbon receptor (AhR) agonists, and estrogenic compounds. At the molecular level, the systems responding to these stressors share common response factors, and evidence exists for cross-talk between them. Biomarkers of exposure to these stressors, cytochrome P4501a (Cyp1a), estrogen receptor alpha (ERα), brain cytochrome P450 aromatase (Cyp19a2 or AromB), and hypoxia inducible factor 1 alpha (Hif-1α) mRNA expression were examined using qRT-PCR simultaneously in embryos of two well studied species, the Atlantic killifish, Fundulus heteroclitus, and the zebrafish Danio rerio. Embryos of both species were exposed to the model Cyp1a inducer β-naphthoflavone (BNF) or 17-β estradiol (E2) under either normoxic or hypoxic (5% oxygen atmosphere) conditions and harvested prior to hatch at 9 days post fertilization (dpf) for the killifish, and 48h post fertilization (hpf) for the zebrafish. BNF significantly induced Cyp1a expression in embryos of both species with killifish embryos being more responsive (700-fold>control) than zebrafish embryos (7-100-fold>control). AromB was also significantly influenced by treatment, but to a lesser extent, with mean expression levels increased by less than two-fold over control values in response to E2, and in one case upregulated by BNF. ERα and Hif-1α were constitutively expressed in embryos of both species, but expression was unaffected by exposure to either BNF or E2. Hypoxic conditions downregulated AromB expression strongly in killifish but not in zebrafish embryos. The impact of hypoxia on expression of other genes in either species was inconsistent, although an interactive effect between hypoxia and BNF on several of the genes evaluated was observed. These data are the first to examine expression patterns of these important environmental response genes together in embryos of two important model fish species. The results support the use of Cyp1a expression as a biomarker of AhR agonists in fish embryos, and indicate that AromB may be more responsive than ERα to estrogenic chemicals at this stage in development. Hif-1α expression was not found to be a good biomarker of hypoxic exposure in either killifish or zebrafish embryos. The interaction observed between BNF and co-exposure to hypoxia warrants further investigation. Finally killifish embryos are generally more sensitive than zebrafish embryos at this stage of development supporting their use in environmental assessments. [Copyright &y& Elsevier]

Abstract: Imposex in female gastropods is a widely documented masculinisation phenomenon in response to tributyltin (TBT) exposure. Although it is generally accepted that imposex is a case of endocrine disruption the underlying mechanisms are controversially discussed with aromatase inhibition and retinoid X receptor (RXR) signalling pathways as two conflicting hypotheses. Hence, we performed injection experiments with the marine dogwhelk Nucella lapillus. As expected TBT induced imposex in all test specimens while the natural RXR ligand 9 cis-retinoic acid did not cause significant effects. Additionally, TBT effects were suppressed if the organotin compound was simultaneously injected with an androgen receptor inhibitor (cyproterone acetate) but not if co-administered with the synthetic RXR antagonist HX531. In contrast, the injection of the RXR agonist HX630 resulted in imposex development in nearly 100% females. Therefore, the results provide evidence for the involvement of the RXR and the androgen signalling pathway. Further investigations are necessary to resolve the biochemical mechanism of imposex development. [Copyright &y& Elsevier]

Abstract: P450 aromatase catalyses the conversion of C19 androgens to C18 estrogens which is thought to be essential for the regulation of the reproductive function. In this study, brain aromatase activity (AA) was measured monthly over a reproductive cycle in wild roach (Rutilus rutilus) sampled in a reference site in Normandy. AA peaked during the breeding season, reaching 35fmolmg−1 min−1 in both male and female fish, and was low during the rest of the year except for a significant rise in October. AA was correlated with ovary maturation (measured either as gonado-somatic index or by histological analysis of the gonads) and plasma sex-steroid levels (11-ketotestosterone in males and 17-β-estradiol in females). Measurements of AA in polluted sites showed that activity was significantly upregulated in sites with fish showing high levels of plasma vitellogenin and large proportion of intersexuality (20–50%) thus suggesting the occurrence of estrogenic compounds and their involvement in AA modulation. [Copyright &y& Elsevier]

Abstract: Produced water (PW), a by-product of the oil-production process, contains large amount of alkylphenols (APs) and other harmful oil compounds. In the last 20 years, there have been increasing concerns regarding the environmental impact of large increases in the amounts of PW released into the North Sea. We have previously shown that low levels of APs can induce disruption of the endocrine and reproductive systems of Atlantic cod (Gadus morhua). The aims of this follow-up study were to: (i) identify the lowest observable effect concentration of APs; (ii) study the effects of exposure to real PW, obtained from a North Sea oil-production platform; and (iii) study the biological mechanism of endocrine disruption in female cod. Fish were fed with feed paste containing several concentrations of four different APs (4-tert-butylphenol, 4-n-pentylphenol, 4-n-hexylphenol and 4-n-heptylphenol) or real PW for 20 weeks throughout the normal period of vitellogenesis in Atlantic cod from October to January. Male and female cod, exposed to AP and PW, were compared to unexposed fish and to fish fed paste containing 17β-oestradiol (E2). Approximately 60% of the females and 96% of the males in the unexposed groups were mature at the end of the experiment. Our results show that exposure to APs and E2 have different effects depending on the developmental stage of the fish. We observed that juvenile females are advanced into puberty and maturation, while gonad development was delayed in both maturing females and males. The AP-exposed groups contained increased numbers of mature females, and significant differences between the untreated group and the AP-treated groups were seen down to a dose of 4μg AP/kg body weight. In the high-dose AP and the E2 exposed groups, all females matured and no juveniles were seen. These results suggest that AP-exposure can affect the timing of the onset of puberty in fish even at extremely low concentrations. Importantly, similar effects were not seen in the fish that were exposed to real PW. [Copyright &y& Elsevier]

Abstract: Ethinyl estradiol is a potent endocrine disrupting compound in fish and ubiquitously present in the aquatic environment. In this study, we exposed adult zebra fish (Danio rerio) males to 0, 5 or 25ng Ethinyl estradiol/L for 14 days and analyzed the effects on non-reproductive behavior. Effects of treatment of the exposed males was shown by vitellogenin induction, while brain aromatase (CYP 19B) activity was not significantly altered. Both concentrations of Ethinyl estradiol significantly altered the behavior in the Novel tank test, where anxiety is determined as the tendency to stay at the bottom when introduced into an unfamiliar environment. The effects were, however, opposite for the two concentrations. Fish that were exposed to 5ng/L had longer latency before upswim, fewer transitions to the upper half and shorter total time spent in the upper half compared with control fish, while 25ng Ethinyl estradiol treatment resulted in shorter latency and more and longer visits to the upper half. The swimming activity of 25, but not 5ng-exposed fish were slightly but significantly reduced, and these fish tended to spend a lot of time at the surface. We also studied the shoaling behavior as the tendency to leave a shoal of littermates trapped behind a Plexiglas barrier at one end of the test tank. The fish treated with Ethinyl estradiol had significantly longer latency before leaving shoal mates and left the shoal fewer times. Further, the fish exposed to 5ng/L also spent significantly less time away from shoal than control fish. Fertilization frequency was higher in males exposed to 5ng/L Ethinyl estradiol when compared with control males, while no spawning was observed after treatment with 25ng/L. The testes from both treatment groups contained a normal distribution of spermatogenesis stages, and no abnormality in testis morphology could be observed. In conclusion, we have observed effects on two behaviors not related to reproduction in zebra fish males after treatment with Ethinyl estradiol, adding to the ecological consequences of contamination of aquatic environments with estrogenic substances. [Copyright &y& Elsevier]

Abstract: Prochloraz is a fungicide known to cause endocrine disruption through effects on the hypothalamic–pituitary–gonadal (HPG) axis. To determine the short-term impacts of prochloraz on gene expression and steroid production, adult female fathead minnows (Pimephales promelas) were exposed to the chemical (0 or 300μg/L) for a time-course of 6, 12 and 24h. Consistent with inhibition of cytochrome P450 17α-hydroxylase/17,20-lyase (CYP17) and aromatase (CYP19), known molecular targets of prochloraz, plasma 17β-estradiol (E2) was reduced within 6h. Ex vivo E2 production was significantly reduced at all time-points, while ex vivo testosterone (T) production remained unchanged. Consistent with the decrease in E2 levels, plasma concentrations of the estrogen-responsive protein vitellogenin were significantly reduced at 24h. Genes coding for CYP19, CYP17, and steroidogenic acute regulatory protein were up-regulated in a compensatory manner in ovaries of the prochloraz-treated fish. In addition to targeted quantitative real-time polymerase chain reaction analyses, a 15k feature fathead minnow microarray was used to determine gene expression profiles in ovaries. From time-point to time-point, the microarray results showed a relatively rapid change in the differentially expressed gene (DEG) profiles associated with the chemical exposure. Functional analysis of the DEGs indicated changes in expression of genes associated with cofactor and coenzyme binding (GO:0048037 and 0050662), fatty acid binding (GO:0005504) and organelle organization and biogenesis (GO:0006996). Overall, the results from this study are consistent with compensation of the fish HPG axis to inhibition of steroidogenesis by prochloraz, and provide further insights into relatively rapid, system-wide, effects of a model chemical stressor on fish. [Copyright &y& Elsevier]

Abstract: Tributyltin (TBT), an antifouling agent, has been implicated in the masculinization of fish species worldwide, but the masculinizing mechanism is not fully understood. We have examined the actions of TBT as an endocrine disruptor in zebrafish (Danio rerio). In HeLa cells transiently co-transfected with plasmid constructs containing the zebrafish estrogen receptors (zfERα, zfERβ1 and zfERβ2) and the zebrafish estrogen response element (zfERE-tk-luc), ethinyl estradiol (EE2) induced luciferase activity 4 to 6-fold and was inhibited by TBT. In HeLa cells transiently co-transfected with the zebrafish androgen receptor (zfAR) and the murine androgen receptor response element (ARE-slp-luc), testosterone induced luciferase activity was not inhibited by TBT. In HeLa cells co-transfected with zfERα, zfERβ1 and zfERβ2 and a plasmid containing zebrafish aromatase (zfCyp19b-luc), TBT inhibited luciferase activity. In zebrafish exposed to 1mg/kg and 5mg/kg TBT in vivo, there was a increase in liver sulfotransferase and a decrease acyl-CoA testosterone acyltransferase activity. Real-time PCR analysis of sexual differentiation markers in fish exposed to TBT in vivo revealed a tissue-specific response. In brain there was increased production of Sox9, Dax1, and SF1 mRNA, an androgenizing effect, while in the liver there was increased production of Dax1, Cyp19a and zfERβ1 mRNA but decreased production of Sox9 mRNA, a feminizing effect. In the gonads there was increased production of zfERα and zfCyp19a mRNA, again a feminizing effect. TBT has an overall masculinizing effect but the masculinizing effect is tempered by a feminizing effect on gene transcription in certain tissues. These results are discussed in the context of TBT as an endocrine disruptor in zebrafish. [Copyright &y& Elsevier]

Abstract: Protein expression changes can be used for detection of biomarkers that can be applied diagnostically to screen chemicals for endocrine modifying activity. In this study, surface enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS) coupled with a short term fish assay was used to investigate changes in plasma protein expression as a means to screen chemicals for androgenic activity. Adult gravid female sheepshead minnows (Cyprinodon variegatus) were placed into separate aquaria for seawater control, ethanol solvent control, and the following androgen agonist treatments at 5.0μg/L: dihydrotestosterone (DHT), methyldihydrotestosterone (MDHT), testosterone (T), methyltestosterone (MT) and trenbolone (TB). Treatments of 0.6μg/L endosulfan and 40μg/L chlorpyrifos (CP) served as non-androgenic negative stressor controls. Test concentrations were maintained using an intermittent flow-through dosing apparatus supplying exposure water at 20L/h. Fish were sampled at 7days, the plasma diluted, processed on weak cation exchange CM10 ProteinChip arrays and analyzed. Spectral processing resulted in 249 individual m/z peak clusters for the androgen exposed fish. Partial least squares-discriminant analysis was used to develop an androgen-responsive model using sample spectra from exposures with DHT and unexposed solvent control fish as the training set. The androgen classification model performed with ≥79% specificity (% true negative) and ≥70% sensitivity (% true positive) for non-aromatizable androgens. The aromatizable androgens T and MT were classified as androgenic with specificities of 42 and 79%, respectively. The reduction in sensitivity observed with T is thought to be caused by its metabolic conversion to an estrogen by aromatase. The results of these studies show diagnostic plasma protein expression models can correctly classify chemicals by their androgenic activity using a combination of high throughput mass spectrometry and multivariate approaches. [Copyright &y& Elsevier]

Abstract: The transcriptional regulator steroidogenic factor 1 (SF-1) and the enzyme cytochrome P450 aromatase (CYP19) play a central role in modulation of a broad range of tissue-specific developmental processes associated with hormone homeostasis that includes differentiation of the central nervous system. SF-1 and CYP19 expression may be targeted by a variety of endocrine disruptive agents prevalent within the environment. In the present study, we cloned and characterized partial sequences for bullfrog (Rana catesbeiana) SF-1 and CYP19 and examined the effects of a 48h exposure to 1 and 100μg/l of the herbicide atrazine (ATZ) and its major metabolite desethylatrazine (DEA), as well as 5ng/l of the estrogenic chemical, 17α-ethynylestradiol (EE2), and 673ng/l of the thyroid hormone, 3,5,3′-triiodothyronine (T3), on SF-1 and CYP19 mRNA abundance in the brains of premetamorphic bullfrog tadpoles. Quantitative RT-PCR analysis showed an increase in CYP19 mRNA following a 48h exposure to EE2 but not T3 while no significant changes in SF-1 transcript levels occurred. We observed a strong positive correlation between CYP19 and SF-1 transcript abundance in the ATZ-exposed animals which was not evident with DEA- or hormone-exposed tadpoles. Our results are intriguing in light of reported behavioral changes in ATZ-exposed frogs and suggest that further research is warranted to examine the relationship and role of CYP19 and SF-1 in amphibian brain development. [Copyright &y& Elsevier]

Abstract: Fluoxetine (FLX) is a pharmaceutical acting as a selective serotonin reuptake inhibitor and is used to treat depression in humans. Fluoxetine and the major active metabolite norfluoxetine (NFLX) are released to aquatic systems via sewage-treatment effluents. They have been found to bioconcentrate in wild fish, raising concerns over potential endocrine disrupting effects. The objective of this study was to determine effects of waterborne FLX, including environmental concentrations, on the reproductive axis in sexually mature male goldfish. We initially cloned the goldfish serotonin transporter to investigate tissue and temporal expression of the serotonin transporter, the FLX target, in order to determine target tissues and sensitive exposure windows. Sexually mature male goldfish, which showed the highest levels of serotonin transporter expression in the neuroendocrine brain, were exposed to FLX at 0.54μg/L and 54μg/L in a 14-d exposure before receiving vehicle or sex pheromone stimulus consisting of either 4.3nM 17,20β-dihydroxy-4-pregnene-3-one (17,20P) or 3nM prostaglandin F2α (PGF2α). Reproductive endpoints assessed included gonadosomatic index, milt volume, and blood levels of the sex steroids testosterone and estradiol. Neuroendocrine function was investigated by measuring blood levels of luteinizing hormone, growth hormone, pituitary gene expression of luteinizing hormone, growth hormone and follicle-stimulating hormone and neuroendocrine brain expression of isotocin and vasotocin. To investigate changes at the gonadal level of the reproductive axis, testicular gene expression of the gonadotropin receptors, both the luteinizing hormone receptor and the follicle-stimulating hormone receptor, were measured as well as expression of the growth hormone receptor. To investigate potential impacts on spermatogenesis, testicular gene expression of the spermatogenesis marker vasa was measured and histological samples of testis were analyzed qualitatively. Estrogen indices were measured by expression and activity analysis of gonadal aromatase, as well as liver expression analysis of the estrogenic marker, esr1. After 14d, basal milt volume significantly decreased at 54μg/L FLX while pheromone-stimulated milt volume decreased at 0.54μg/L and 54μg/L FLX. Fluoxetine (54μg/L) inhibited both basal and pheromone-stimulated testosterone levels. Significant concentration-dependent reductions in follicle-stimulating hormone and isotocin expression were observed with FLX in the 17,20P- and PGF2α-stimulated groups, respectively. Estradiol levels and expression of esr1 concentration-dependently increased with FLX. This study demonstrates that FLX disrupts reproductive physiology of male fish at environmentally relevant concentrations, and potential mechanisms are discussed. [Copyright &y& Elsevier]

Abstract: Until now, studies dedicated to the actions of endocrine disrupting chemicals (EDCs) on the reproductive axis have been concerned with their effects at the gonadal level leaving their actions on neuroendocrine circuits controlling reproduction virtually unexplored. In vertebrates, gonadotropin-releasing hormone (GnRH) is the key factor controlling the activity of the reproductive axis. The development and functioning of GnRH neurons are finely tuned by a series of factors, notably sex steroids, making these neurons potential targets of EDCs, notably in aquatic species. By means of immunohistochemistry, we examined the effects of low levels of ethinylestradiol (EE2 0.02nM, 0.1nM, 0.5nM), a potent synthetic estrogen, on early development (at 5, 10, 20, 30 days post-fertilization) of the forebrain GnRH neurons in a model fish species, the zebrafish (Danio rerio). In parallel, the ER-regulated expression of cytochrome P450 aromatase B (AroB) protein, which is encoded by the cyp19a1b gene, was precisely mapped at the brain and pituitary levels in developing control and EE2-exposed zebrafish. We show that EE2 disrupts the ontogeny of GnRH system by inducing an increase in the number of GnRH-ir neurons and GnRH fibers based on their immunoreactivity as well as a decrease in the size of the GnRH-ir soma and a modification of the migration profile of GnRH-ir neurons. Furthermore, we report a spectacular dose and time-dependent induction of AroB expression in radial glial cells of the developing brain further illustrating the extreme sensitivity of AroB to xenoestrogen and the relevance of AroB as biomarker of xenoestrogen effects on the central nervous system. Collectively, these original and relevant observations highlight the sensitivity of GnRH and AroB to a synthetic estrogen during embryogenesis. These data reinforce the need to further study the mechanisms underlying EDC effects on key neuroendocrine circuits involved in reproduction and brain development of vertebrates. [Copyright &y& Elsevier]

Abstract: The mechanisms by which organotin compounds produce modulations of the endocrine systems and other biological responses are not fully understood. In this study, juvenile salmon were force-fed diet containing TBT (0: solvent control, 0.1, 1 and 10mg/kg fish) for 72h. Subsequently, fish exposed to solvent control and 10mg TBT were exposed to waterborne concentration (200μg/l) of the adenylate cyclase (AC) stimulator, forskolin for 2 and 4h. The overall aim of the study was to explore whether TBT endocrine disruptive effects involve second messenger activation. Liver was sampled from individual fish (n =8) at the end of the exposures. The transcription patterns of peroxisome proliferator-activated receptor (PPAR) isotype and acyl-coenzyme A oxidase 1 (ACOX1), aromatase isoform, estrogen receptor-α (ERα), pregnane X receptor (PXR), CYP3A and glutathione S-transferase (GST) genes were measured by quantitative polymerase chain reaction (qPCR). Our data showed a consistent increase in PPARα, PPARβ and PPARγ mRNA and protein expression after TBT exposure that were inversely correlated with ACOX1 mRNA levels. Forskolin produced PPAR isotype-specific mRNA and protein effects that were modulated by TBT. ACOX1 expression was decreased (at 2h) and increased (at 4h) by forskolin and the presence of TBT potentiated these effects. TBT apparently increased mRNA and protein levels of cyp19a, compared to the solvent control, whereas cyp19b mRNA levels were unaffected by TBT treatment. Combined TBT and forskolin exposure produced respective decrease and increase of mRNA levels of cyp19a and cyp19b, compared with control. TBT decreased ERα mRNA at low dose (1mg/kg) and forskolin exposure alone produced a consistent decrease of ERα mRNA levels that were not affected by the presence of TBT. Interestingly, PXR and CYP3A mRNA levels were differentially affected, either decreased or increased, after exposure to TBT and forskolin, singly and also in combination. GST mRNA was increased by TBT exposure. Exposure to forskolin alone increased GST expression with time, and combined exposure with TBT potentiated these respective effects. Overall, the present study demonstrates multiple biological effects of TBT given singly or in combination with cAMP activator. There are no studies known to us that have evaluated the endocrine disruptive effects of TBT in the presence of a second messenger activator, and our data suggest that TBT may exert endocrine, biotransformation and lipid peroxidative effects through modulation of cAMP/PKA second messenger signaling with overt physiological consequences. [Copyright &y& Elsevier]

Abstract: Despite frequent detection of ibuprofen in aquatic environments, the hazards associated with long-term exposure to ibuprofen have seldom been investigated. Ibuprofen is suspected of influencing sex steroid hormones through steroidogenic pathways in both vertebrates and invertebrates. In this study, the effect of ibuprofen on sex hormone balance and the associated mechanisms was investigated in vitro by use of H295R cells. We also conducted chronic toxicity tests using freshwater fish, Oryzias latipes, and two freshwater cladocerans, Daphnia magna and Moina macrocopa, for up to 144 and 21d of exposure, respectively. Ibuprofen exposure increased 17β-estradiol (E2) production and aromatase activity in H295R cells. Testosterone (T) production decreased in a dose-dependent manner. For D. magna, the 48h immobilization EC50 was 51.4mg/L and the 21d reproduction NOEC was <1.23mg/L; for M. macrocopa, the 48h immobilization EC50 was 72.6mg/L and the 7d reproduction NOEC was 25mg/L. For O. latipes, 120d survival NOEC was 0.0001mg/L. In addition, ibuprofen affected several endpoints related to reproduction of the fish, including induction of vitellogenin in male fish, fewer broods per pair, and more eggs per brood. Parental exposure to as low as 0.0001mg/L ibuprofen delayed hatching of eggs even when they were transferred to and cultured in clean water. Delayed hatching is environmentally relevant because this may increase the risk of being predated. For O. latipes, the acute-to-chronic ratio of ibuprofen was estimated to be greater than 1000. Overall, relatively high acute-to-chronic ratio and observation of reproduction damage in medaka fish at environmentally relevant ranges of ibuprofen warrant the need for further studies to elucidate potential ecological consequences of ibuprofen contamination in the aquatic environment. [Copyright &y& Elsevier]

Abstract: 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 1.00mgL−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. [Copyright &y& Elsevier]

Abstract: We evaluated plasma testosterone (T) and 17β-estradiol (E2) levels and ovarian aromatase CYP19a gene expression following a single intraperitoneal injection of Chilean pulp and paper mill effluent extracts into juvenile triploid rainbow trout. Fish injected with untreated effluent extracts had increased plasma T after 4 days, while plasma E2 concentration was increased in fish injected with both primary and secondary treated effluent extracts at the same sampling period. Ovarian CYP19a gene expression as measured by qRT-PCR was significantly induced in fish injected with the untreated, primary and secondary treated pulp and paper mill effluent extracts. Similar induction of CYP19a expression was found in fish injected with the androgens androstenedione (ADD) and T. A Principal Component Analysis (PCA) was conducted in order to identify structure in relationships between all measured variables and identifying which factors were most responsible for the variance observed within the plasma steroid levels, upregulation of ovarian CYP19a gene expression and the final estrogenic effect of increased plasma VTG levels. This analysis indicated a cluster correlation between plasma T levels and CYP19a gene expression (Factor 1, explaining 27.2% of total variance), a cluster including condition factor and liver somatic index (Factor 2, explaining 17.3%) and an additional cluster including plasma E2 and vitellogenin levels (Factor 3, explaining an additional 15.8%). The present results indicate that Chilean pulp and paper mill effluent extracts cause estrogenic effects in triploid rainbow trout. These effects could be related to the compounds present in the effluent that act as estrogen receptor agonists, or that induce changes leading to increased amounts of endogenous estrogens, reflected by increased E2 levels and induced aromatase expression/activity. [Copyright &y& Elsevier]

Abstract: Clotrimazole is a pharmaceutical used for treatment of fungal infections. It has been found in surface waters outside municipal wastewater treatment plants but data are scarce regarding its effects on aquatic organisms. It is known that clotrimazole and other imidazole fungicides are inhibitors of the enzyme aromatase (CYP 19). Aromatase converts androgens into estrogens and is suggested to be involved in the sex differentiation in amphibians. The aim of the present study was to evaluate effects of larval exposure to clotrimazole on aromatase activity in brain and gonads, and on gonadal differentiation in Xenopus tropicalis frogs. Another purpose was to determine if larval exposure to ethynylestradiol (EE2), at a concentration known to cause male-to-female sex reversal, affects aromatase activity in brain and gonads during gonadal differentiation. Tadpoles were exposed from shortly after hatching (Nieuwkoop and Faber developmental stages 47–48) until complete metamorphosis (NF stage 66) to 6, 41, and 375nM clotrimazole or 100nM (nominal) EE2. Aromatase activity was measured in the brain and gonad/kidney complex of tadpoles during gonadal differentiation (NF stage 56) and, in the clotrimazole experiment, also at metamorphosis. In clotrimazole-exposed tadpoles gonadal aromatase activity increased over exposure time in the 41 and 375nM groups but did not differ significantly from the control group. Gonadal aromatase activity was increased in both sexes exposed to 41 and 375nM clotrimazole at metamorphosis. Brain aromatase activity was decreased in tadpoles (NF stage 56) exposed to 375nM clotrimazole, but at metamorphosis no differences were seen between groups or between sexes. No effects of clotrimazole on sex ratio or gonadal histology were noted at completed metamorphosis. EE2-exposed tadpoles had a slightly decreased gonadal aromatase activity, though not significantly different from control group, and there was no effect of EE2 on brain aromatase activity. All EE2-exposed tadpoles developed ovaries. These findings indicate that estrogen-induced ovarian differentiation is not paralleled by increased gonadal aromatase activity in X. tropicalis. Further studies are needed, especially on developmental reproductive toxicity, to assess the risk for endocrine disruption in wild amphibians posed by clotrimazole and other imidazole fungicides. [Copyright &y& Elsevier]

Abstract: Aromatase is a steroidogenic enzyme that catalyzes the conversion of androgens to estrogens in vertebrates. Modulation of this enzyme''s activity by xenobiotic exposure has been shown to adversely affect gonad differentiation in a number of diverse species. We hypothesized that exposure to the aromatase inhibitor, fadrozole, during the larval development of the tropical clawed frog, Xenopus tropicalis, would result in masculinization of the developing female gonad. Tadpoles were exposed to fadrozole at nominal concentrations from 1 to 64μg/L in a flow-through system from <24h post-fertilization (Nieuwkoop Faber (NF) stage 15–20) to metamorphosis (NF stage 66). At metamorphosis, morphologically examined gonads indicated complete masculinization of all tadpoles at concentrations of 16μg/L and above and a significant bias in sex ratio towards males at concentrations of 1μg/L and above. No effects on time to metamorphosis, body mass, or body length were observed. A random subsample of frogs was raised to reproductive maturity (39 weeks post-fertilization) in control water. All frogs exposed as tadpoles to 16μg/L fadrozole or greater possessed testes at sexual maturity. Intersexed gonads characterized by the presence of both testicular and ovarian tissue were observed in 12% of frogs in the 4μg/L treatment. No differences in estradiol, testosterone, or vitellogenin plasma concentrations were observed in exposed males or females compared to controls. Females in the 4μg/L treatment possessed a significantly greater percentage of pre-vitellogenic oocytes than controls and were significantly smaller in body mass. No differences in sperm counts were observed in exposed males compared to controls. Results from this study demonstrate that larval exposure to an aromatase inhibitor can result in the complete masculinization of female gonads. These masculinized females are phenotypically indistinguishable from normal males at adulthood. Lower levels of aromatase inhibition resulted in intersexed gonads and possible female reproductive impairment at adulthood. These results indicate that exposure of amphibians to xenobiotics capable of inhibiting aromatase would result in adverse reproductive consequences. [Copyright &y& Elsevier]

• Aromatase activity is a potential biomarker of the effect of endocrine disruptors. • The greatest effect on Cyp19a expression in zebrafish was observed at 10 μg/L BPA. • Theca cells in ovary and radial glial cells in brain are the targets of BPA action. • BPA toxic effect could lead to the development of reproductive dysfunction. • BPA toxic effect could impair self-repair processes in the brain. Bisphenol A (BPA) is used to produce plastic and plastic derived products in multitude of daily utensils, being one of the industrial compounds most widely used. This endocrine disrupting chemical (EDCs) is a well-known environmental pollutant released into the aquatic environment from industrial wastewater, sewage sludge or landfill leachate. Aromatases are considered potential targets of EDCs with characteristics that make them suitable biomarkers of exposure to their effects. The main objective of our study was to evaluate the expression of cyp19a aromatase as a toxicological endpoint after BPA exposure through the identification and assessment of alterations of the main cells responsible for cyp19a1a and cyp19a1b expression in the zebrafish ovary and brain using different concentrations of BPA in water. Immunohistochemistry was used to analyze the expression of these enzymes in female zebrafish exposed and not exposed to different concentrations of BPA (1, 10, 100 and 1000 μg / L) in water (n = 6/group) for 14 days. The results obtained in this study showed that the cyp19a aromatase system, involved in the synthesis of steroid compounds, is specially located in distinct oocyte stages in the ovary (cyp19a1a) and in radial glial cells of the brain (cyp19a1b). An overexpression of these aromatases was observed after BPA exposure in zebrafish, peaking from a concentration of 10 µg/L and showing to be good biomarkers of exposure to identify the early effects of low BPA concentrations. To our knowledge, this study is the first to localize and quantify the expression of cyp19a1a and cyp19a1b in the cells of brain and ovary after fish exposure to different BPA concentrations in water. [ABSTRACT FROM AUTHOR]

Abstract: The higher molecular weight polycyclic aromatic hydrocarbons (PAHs) such as benzo(a)pyrene (BaP) are typically associated with genotoxicity, however, newer evidence suggests that these compounds may also act as endocrine system disruptors. We hypothesized that altered expression of the P450 enzyme aromatase genes could be a target for reproductive or developmental dysfunction caused by BaP exposure. Aromatase is at least partially responsible for estrogen homeostasis by converting androgens into estrogens. In fish, there are two isoforms of aromatase, a predominantly ovarian form, CYP19A1, and a brain form, CYP19A2. CYP19 mRNA expression was measured following BaP exposure (0, 10, 100μg/L waterborne for 10 or 15 days) in Fundulus adults, juveniles and embryos by in situ hybridization. The CYP19A1 expression was significantly decreased after BaP exposure in the 3-month-old Fundulus immature oocytes, but BaP did not affect CYP19A1 expression at any stage in adult oocytes. In embryo brains, BaP significantly decreased CYP19A2 compared to controls by 3.6-fold at 14 days post-fertilization. In adults, CYP19A2 expression was decreased significantly in the pituitary and hypothalamus (81% and 85% of controls, respectively). Promoter regions of Fundulus CYP19s were cloned, and putative response elements in the CYP19A1 and CYP19A2 promoters such as CRE, AhR and ERE may be involved in BaP-mediated changes in CYP19 expression. In order to compare the mechanism of BaP-mediated inhibition with that of a known aromatase inhibitor, fish were also exposed to fadrozole (20 and 100μg/L). Fadrozole did not significantly decrease the mRNA expression in embryos or adult Fundulus. However, aromatase enzyme activity was significantly decreased in adult ovary and brain tissues. These studies provide a greater molecular understanding of the mechanisms of action of BaP and its potential to impact reproduction or development. [Copyright &y& Elsevier]

Abstract: There is a growing international concern that commonly used environmental contaminants have the potential to disrupt the development and functioning of the reproductive system in amphibians. One such chemical of interests is the herbicide atrazine. Effects of atrazine on sex differentiation were studied using wild-type Xenopus laevis tadpoles and all-ZZ male cohorts of X. laevis tadpoles, produced by mating wild-type ZZ male to sex-reversed ZZ male (female phenotype). Stage 49 tadpoles were exposed to 0.1–100ppb atrazine or 0.27ppb (1nM) 17β-estradiol (E2) until all larvae completed metamorphosis (stage 66). Metamorphosis, gonadal morphology and histology, CYP19 (P450 aromatase) mRNA induction, and hepatic vitellogenin (VTG) induction were investigated. Effects of atrazine on VTG-induction were also assessed in vitro in primary-cultured X. laevis hepatocytes. Atrazine had no effect on metamorphosis of developing wild-type or all-male X. laevis larvae. Statistical increase in female ratios was observed in 10 and 100ppb atrazine groups in comparison with control group. While no hermaphroditic froglet was observed in all atrazine groups. In ZZ males, sex reversal was induced by 0.27ppb E2, but not by atrazine at concentrations of 0.1 and 1.0ppb. In addition, neither P450 aromatase mRNA in the gonad nor hepatic VTG were induced by atrazine. Furthermore, VTG was not induced by 1000ppb atrazine in primary-cultured hepatocytes. Our results indicate that female ratios in developing X. laevis tadpoles were increased by 10 and 100ppb atrazine under the present experimental conditions. While the other endpoints showed no effect in the range of 0.1–100ppb atrazine. These results suggest that effect of atrazine on sexual differentiation was not caused by estrogenic action and has no induction ability of P450 aromatase gene in gonad. [Copyright &y& Elsevier]

Abstract: Androgen metabolism (androstenedione and testosterone) has been assessed in the digestive tube and gonads of the echinoderm Paracentrotus lividus exposed to different concentrations of the biocide triphenyltin (TPT) in a semi-static water regime for 4 weeks. Key enzymatic activities involved in both synthesis and metabolism of androgens, namely 17β-hydroxysteroid dehydrogenases (17β-HSDs), 3β-HSDs, 5α-reductases, P450-aromatase, palmitoyl-CoA:testosterone acyltransferases (ATAT) and testosterone sulfotransferases (SULT), were investigated in digestive tube and/or gonads of control and TPT-exposed specimens in an attempt to see whether androgen metabolism was altered by exposure. In agreement with previous data for vertebrates, exposure to TPT led to a concentration dependent decrease of P450-aromatase that was statistically significant at the highest TPT concentration tested (225ng/L). Additionally, increased metabolism of testosterone to form dihydrotestosterone (DHT) and 5α-androstane-3β,17β-diol was observed, suggesting increased 5α-reductase activity in the gonads of TPT-exposed individuals. Interestingly, exposure to TPT induced testosterone conjugating activities in organisms exposed to medium (SULT) and high (ATAT and SULT) TPT concentrations. Despite the changes of androgen metabolizing enzymes, testosterone levels in gonads remained rather stable. In contrast, an increase in testosterone and a concomitant decrease in estradiol were observed in the coelomic fluid of TPT-exposed organisms. Overall, the data indicate the ability of TPT to modulate androgen metabolism and circulating steroid levels in P. lividus and suggest the existence of regulatory mechanisms to maintain stable endogenous levels of testosterone in gonads. This study also contributes to a better knowledge of echinoderm endocrinology. [Copyright &y& Elsevier]

Abstract: Cytochrome P450 aromatase (CYP19) plays an important role in steroid homoeostasis by converting androgens to estrogens. To evaluate the effects of benzo(a)pyrene (BaP), a model carcinogenic PAH and AhR ligand, on aromatase mRNA expression and enzyme activity, adult Fundulus were exposed to water-borne BaP (1 and 10μg/L) for 15 days, and embryos were exposed to 10μg/L for 10 days. Effects of BaP were examined by tissue, gender, and season in adults. Constitutively, the sexes did not have significantly different CYP19A2 mRNA levels, however females had higher brain aromatase activity. Female control killifish had more than 700-fold more CYP19A1 mRNA in their gonads compared to males. Within brain tissue of both sexes, there was 100-fold more CYP19A2 mRNA compared to CYP19A1. In ovary, CYP19A1 predominated by approximately 30-fold over the CYP19A2, but in testis there was relatively more CYP19A2. In embryos there was ∼5-fold higher CYP19A2 expression. Due to high inter-individual variability, a significant effect of BaP treatment by gender, season or age was not observed for either aromatase mRNA. However, ovarian aromatase activity was significantly decreased by 10μg/L BaP, while female brain activity was increased following winter exposure. These findings suggest that the aromatase enzyme is a potential target for disruption of fish developmental and reproductive physiology by BaP. [Copyright &y& Elsevier]

Abstract: The molecular basis for vitellogenin (Vtg) and Zr-protein gene (and protein) expression shows that the Vtg and Zr-protein gene activations are receptor-mediated responses that are ligand structure-dependent interactions with ER, probably involving all isoforms, in addition to other co-activators. In addition to their effect as direct agonist to the ERs, the endocrine-disrupting effects of environmental chemicals are sometimes interpreted as interference with steroidogenesis and with the steroidal regulation of the normal development and function of the male and female reproductive tracts. In vertebrates, the cytochrome P450 aromatase (P450arom) is a crucial steroidogenic enzyme catalyzing the final step in the conversion of androgens to estrogens. The present study was undertaken to investigate the effects of nonylphenol on brain and liver ER and P450arom isotypes gene expressions as sensitive biomarker of effect. Fish were exposed to static and continuous waterborne nonylphenol at 5, 15 and 50μg/L. Blood, brain and liver samples were collected after 0 (control), 3 and 7 days post-exposure. Plasma levels of Vtg and Zr-proteins were analyzed using immunoblotting and enzyme linked immunosorbent assay (ELISA) methods. A quantitative RT-PCR method was used to analyse gene expression patterns using cloned plasmid containing amplicons of interest as standards. Our data demonstrate that both ER and P450arom gene isotypes showed differential organ-specific, nonylphenol concentration- and time-dependent expression patterns after exposure to environmental relevant concentrations of nonylphenol. Liver and plasma Vtg and Zr-protein gene and protein expression showed a concentration- and time-dependent induction at day 3 and 7 post-exposure. This is the first study evaluating, in parallel, the ERs, P450aroms and protein xenoestrogen biomarkers of effect in any fish species or lower vertebrate. Therefore, the present study shows that the brain and hepatic ER and P450arom isotypes evaluated using state-of-the-art molecular approaches are sensitive xenoestrogen biomarkers of effect. These responses should be evaluated in parallel with plasma protein biomarkers such as Vtg and Zr-proteins. [Copyright &y& Elsevier]

Abstract: The triazine herbicide atrazine has been hypothesized to disrupt sexual development in frogs by up-regulating aromatase activity, resulting in greater estradiol (E2) concentrations and causing feminization in males. The goal of this study was to collect native ranid frogs from atrazine-exposed ponds and determine whether relationships exist between measured atrazine concentrations and the gonadosomatic index (GSI), plasma concentrations of testosterone (T), E2 or 11-ketotestosterone (KT), or with aromatase activity. In the summer of 2002 and 2003, adult and juvenile green frogs (Rana clamitans), bullfrogs (R. catesbeiana) and Northern leopard frogs (R. pipiens) were collected from areas with extensive corn cultivation and areas where there was little agricultural activity in south-central Michigan. Atrazine concentrations were below the limit of quantification at non-agricultural sites. Atrazine concentrations did not exceed 2μg/L at most agricultural sites, but a concentration of 250μg atrazine/L was measured in one sample from one site in 2002. Plasma steroid concentrations varied among locations. Aromatase activity was measurable in less than 11% of testes in adult males, and in less than 4% of testes in juvenile males. Median aromatase activities in ovaries of adult females ranged from 3 to 245pmol/h/mg protein, and maximum activities were 2.5-fold greater in juveniles than in adults. Atrazine concentrations were not significantly correlated with any of the parameters measured in this study. These results indicate that atrazine does not up-regulate aromatase in green frogs in the wild, and does not appear to affect plasma steroid hormone concentrations. [Copyright &y& Elsevier]

Abstract: Short-term effects of methyltestosterone (MT) on the endocrine system of adult male zebrafish (Danio rerio) were examined. Males were exposed to 0, 4.5, 6.6, 8.5, 19.8, 35.9, 62.3ng MT/l and ethinylestradiol (EE2) (26.4ng/l) for 7 days. Several physiological endpoints that may be affected by endocrine disrupters were analysed, specifically vitellogenin (VTG) concentration, estradiol (E2), testosterone (T), and 11-ketotestosterone (KT) content, brain aromatase activity and gene expression of CYP19A1 and CYP19A2 in the testis. Exposure to the lowest MT concentration (4.5ng MT/l), and the EE2 increased the concentration of VTG significantly compared to solvent control group. Exposure to higher concentrations of MT did not increase VTG levels. Endogenous KT and T levels decreased significantly in a concentration-dependent manner in response to the MT exposure and the lowest effective concentrations were 6.4 and 8.5ng MT/l, respectively. The levels of KT and T were also significantly suppressed by EE2 when compared to the solvent control group. Significant decreases in endogenous E2 levels were found in some MT groups but it was not possible to distinguish a simple concentration–response relationship. No effects of MT or EE2 on the brain aromatase activity or on testicular gene expression of CYP19A1 and CYP19A2 were detected. The results show that androgens such as MT can act as endocrine disrupters even at very low concentrations. [Copyright &y& Elsevier]

Abstract: There is growing evidence that some chemicals present in the environment have the capacity to inhibit, or potentially induce, aromatase activity. This study compared aromatase activities and isoform-specific mRNA expression in brain and ovary tissue from non-exposed fathead minnows representing three different ages and stages of reproductive activity, and from fathead minnows exposed to the aromatase inhibitor fadrozole for 7d. The goal was to determine whether measures of a single aromatase endpoint in either brain or ovary tissue would be sufficient to understand and predict system-wide effects of endocrine disrupting chemicals on aromatase activity and transcript levels. Aromatase activity in the ovary, but not brain, varied significantly with age/reproductive category, with adults held in non-reproductive conditions showing significantly lower activity than juveniles and reproductively-active adults. Significant correlations between isoform-specific transcript levels and aromatase activity were observed for ovary tissue, but those relationships were not robust for all age/reproductive categories, nor were they sustained in fadrozole-treated fish. In vitro, fadrozole inhibited the aromatase activity of brain and ovary post-mitochondrial supernatants with similar potency (IC50s=8.82±1.58 and 6.93±0.80μM for brain and ovary, respectively), despite large differences in the magnitude of activity. In vivo, fadrozole altered aromatase activity and isoform-specific transcript levels in both brain and ovary tissue, but concentration-response relationships were different for each tissue. Aromatase activity and P450aromB mRNA expression in brain showed a dose-dependent decrease at concentrations greater than 5.55μg/L. In contrast, ovary activity showed an inverted U-shaped concentration-response consistent with the interplay between increased P450aromA transcript levels in ovary and competitive inhibition of the aromatase enzyme. As a whole, results of this study did not reveal any robust correlations between brain and ovary aromatase activity and/or isoform-specific mRNA expression. However, they were consistent with the current body of evidence related to teleost aromatase regulation, suggesting that increased understanding of the biology of aromatase may facilitate system-wide understanding of effects on aromatase based on relatively few measured endpoints. [Copyright &y& Elsevier]

Abstract: We have previously found that leachate from a Swedish refuse dump caused toxicological effects, including endocrine disruption and reproductive failures, in feral female perch (Perca fluviatilis) from Molnbyggen and in brook trout (Salvelinus fontinalis) from Vadbäcken. This raised concerns that leachate-induced toxicity might affect fish in other leachate-contaminated lakes. This study looks at female perch from three different regions in Sweden, focusing on toxicological biomarkers (skin lesions, liver-somatic index (LSI), ethoxyresorufin O-deethylase (EROD) activity and DNA adducts) and reproductive biomarkers (number of mature females, gonadosomatic index (GSI), aromatase (P450arom), progesterone, 17α-hydroxyprogesterone (17α-OHP), testosterone (T) and 17β-estradiol (E2)). Five out of seven contaminated locations had lower numbers of mature females and most of them exhibited higher frequencies of fin erosion relative to their comparable reference sites. Females from Lake Nedre Vättern exhibited these effects, as well as body sores, high EROD activity, high levels of DNA adducts in the liver and the intestinal mucosa, low GSI and low plasma levels of T and E2, similar to the anti-estrogenic effects earlier found in SM perch from Molnbyggen in 1997 and in brook trout from Vadbäcken. No effects on LSI, GSI, aromatase, or circulating steroids were found in mature females from Molnbyggen in this study. This indicated less leachate-contamination, but low numbers of mature females in Lake Siljan, at the sewage treatment plant which now processes the leachate that earlier contaminated Molnbyggen and Vadbäcken, suggested insufficient sewage treatment at this site. All females in Lake Simshyttsjön were mature, and their high GSI and LSI, low EROD activity, high level of 17α-OHP and low level of T, suggest an estrogenic rather than an anti-estrogenic effect. The results show that our earlier findings in Molnbyggen and Vadbäcken are not common, but neither are they unique. This study also shows that refuse dumps should be considered as potential point-sources for environmental pollutants, and that uncontrolled leachate-contamination of lakes and freshwater reservoirs could be a serious environmental hazard for both wildlife and humans. [Copyright &y& Elsevier]

Abstract: Mussels (Mytilus galloaprovincialis) were exposed to different concentrations of estradiol (20, 200, and 2000ng/L) in a semi-static regime (1-day dosing intervals) for up to 7 days in an attempt to see how mussels deal with exogenous estrogenic compounds. Whole tissue free-estradiol levels were only significantly elevated at the high exposure dose, whereas total-estradiol (free+esterified) sharply increased in a dose-dependent manner, from 2ng/g in controls to 258ng/g at the high exposure group. Neither free nor esterified testosterone levels showed significant differences between control and exposed organisms. The results suggest the existence of mechanisms that allow mussels to maintain their hormonal levels stable, with the exception of the high exposure dose, and the important role that fatty acid esterification, e.g. palmitoyl-CoA:estradiol acyltransferases, may play within those mechanisms. Additionally, the activity of 17β-hydroxysteroid dehydrogenase (17β-HSD), 5α-reductase, P450-aromatase, and estradiol-sulfotransferases were investigated in digestive gland microsomal and cytosolic fractions. All these activities were differently affected by estradiol exposure. Overall, the study contributes to the better knowledge of molluscan endocrinology, and defines new mechanisms of regulation of free steroid-levels in mussels. [Copyright &y& Elsevier]

Abstract: The ultrastructure of testicular cells of adult male African clawed frogs (Xenopus laevis) exposed to either estradiol (0.1μg/L) or 2-chloro-4-ethylamino-6-isopropyl-amino-s-triazine (atrazine; 10 or 100μg/L) was examined by electron microscopy and compared to plasma concentrations of the steroid hormones, testosterone (T) and estradiol (E2), testicular aromatase activity and gonad growth expressed as the gonado-somatic index (GSI). Exposure to E2 caused significant changes both at the sub-cellular and biochemical levels. Exposure to E2 resulted in significantly fewer sperm cells, inhibition of meiotic division of germ cells, more lipid droplets that are storage compartments for the sex steroid hormone precursor cholesterol, and lesser plasma T concentrations. Although not statistically significant, frogs exposed to E2 had slightly smaller GSI values. These results may be indicative of an inhibition of gonad growth and disrupted germ cell development by E2. Concentrations of E2 in plasma were greater in frogs exposed to E2 in water. Exposure to neither concentration of atrazine caused effects on germ cell development, testicular aromatase activity or plasma hormone concentrations. These results suggest that atrazine does not affect testicular function. In contrast, exposure of male X. laevis to E2 led to sub-cellular events that are indicative of disruption of testicular development, and demasculinization processes (decrease of androgen hormone titers). These results indicate that atrazine does not cause responses that are similar to those caused by exposure to E2. [Copyright &y& Elsevier]

Abstract: Populations of killifish (Fundulus heteroclitus) persist in many different highly polluted environment indicative of adaptation or tolerance. In this study, we sought to determine whether long term, multigenerational exposures to environmental contaminants has affected reproductively relevant genes and biological processes. A homology cloning strategy was used to isolate the killifish cytochrome P450 aromatase (P450arom, estrogen synthetase) cDNAs. Consistent with previous fish studies, killifish were found to have two P450arom forms, which segregated into A- and B-gene clades and were differentially expressed in brain (B≫A) and ovary (A≫B). Comparison of killifish from highly polluted (New Bedford Harbor, NBH) and unpolluted (Scorton Creek, SC) environments revealed no site-related differences in P450arom coding sequences or in overall tissue distribution patterns. As measured by real-time quantitative PCR (QPCR) analysis, however, P450arormB (a known marker of estrogen effect) was approximately two-fold higher in the brain of NBH than of SC fish, a difference seen in reproductively active and inactive males and females. Providing further evidence of exposure to estrogen-like pollutants or metabolites in NBH, vitellogenin (vtg) mRNA and protein were elevated in seasonally active and inactive males, and in reproductively inactive females, when compared to SC fish. By contrast, during the period of reproductive activity, NBH females had a lower gonadosomatic index, lower plasma estrogen, a decreased hepatosomatic index, and reduced vtg expression as compared to SC females, indicating that the female hypothalamic-pituitary-gonadal (HPG)-liver axis is impaired in the polluted environment. As measured by a decrease in plasma androgen (but not GSI), the male HPG axis was impaired in reproductively active NBH versus SC fish. In agreement with reports that NBH killifish are resistant to dioxin-like chemicals (DLC) that activate arylhydrocarbon receptor (AhR) signaling, ovarian P450aromA (a marker of dioxin-like effect in zebrafish embryos) did not differ in SC and NBH fish. In conclusion, the killifish population at the NBH Superfund site maintains a level of reproductive competence in the face of evidence of exposure to estrogen-like pollutants and endocrine disruption. [Copyright &y& Elsevier]

Screening assays have been successfully developed for the detection of (anti-)oestrogenic substances in several fish species, including the fathead minnow (Pimephales promelas). Previous work suggested that pre-spawning adult fathead minnows might be an appropriate life-stage for developing a screen to detect endocrine active substances (EASs). Pre-spawning adult fathead minnows, in which their phenotypic sex could be determined, were exposed in flow-through systems to three reference substances for 21 days, at 25°C. Male and female fish, held in separate tanks, were exposed to dihydrotestosterone (DHT, androgen), flutamide (anti-androgen) and fadrozole (aromatase inhibitor). Nominal (mean measured) concentrations for DHT were 10 (6.0), 32 (6.1) and 100 (8.6)μgl-1, for flutamide, 100 (95.3), 320 (320.4) and 1000 (938.6)μg l-1 and for fadrozole, 25 (24.8), 50 (51.7) and 100 (95.5)μgl-1. After 14 and 21 days exposure, fish were evaluated for growth, secondary sexual characteristics (SSCs, number and prominence of nuptial tubercles), gonadosomatic index (GSI) and plasma vitellogenin (VTG) concentrations. Development of nuptial tubercles was sensitive to both DHT and flutamide exposure. Exposure to DHT significantly increased the number of nuptial tubercles (male characteristic) in both males (more abundant) and females, after 14 days. Flutamide (938.6μgl-1, day 21) significantly reduced nuptial tubercle number in male fish. Fadrozole significantly inhibited ovarian growth (lower GSI) and significantly induced testis growth (51.7 and 95.5μgl-1), after 21 days. Plasma VTG concentrations were significantly elevated in male fish (6.1 and 8.6μgl-1), but inhibited in female fish (6.0μgl-1), exposed to DHT. Flutamide had no effect on plasma VTG in male fish, but significantly induced VTG in female fish, after 21 days. Fadrozole significantly inhibited VTG in females and induced VTG synthesis in males, at day 21. These results show that SSCs, GSI and plasma VTG concentrations can be used in pre-spawning adult fathead minnows to screen for a range of classes of EASs. This work complements other published studies in supporting the current OECD effort towards validating a 21 days non-spawning fish screening assay for assessing (anti-)oestrogens, aromatase inhibitors and (anti-)androgens. [Copyright &y& Elsevier]

This study investigated whether gonadal sex differentiation of zebrafish (Danio rerio) is susceptible to compounds that interfere with cytochrome P450 aromatase (P450arom). Treatment of zebrafish during the period of gonadal differentiation with either the non-steroidal aromatase inhibitor fadrozole or 17α-methyltestosterone (MT) changed gonad morphological differentiation and altered the pattern of P450arom gene (CYP19) expression. Application of fadrozole (500 μg/g of food) between days 35 and 71 post-fertilisation (pf) resulted in 100% masculinisation, i.e. the gonads of all individuals examined (n=40) showed testicular morphology. At the same time, fadrozole treatment suppressed gonadal CYP19A (gonad-derived CYP19 gene) mRNA expression, as assessed by means of semi-quantitative RT-PCR. After termination of fadrozole treatment at day 71 pf and subsequent rearing of zebrafish under control conditions until 161 days pf, the gonads of all individuals still displayed testicular morphology. Gonadal CYP19A expression, however, showed a dimorphic pattern, with 14 out of 22 individuals having low CYP19A mRNA levels similar to those found in testes of control fish, while eight fish showed high, ovary-like levels of gonadal CYP19A mRNA. MT treatment (10 μg/l) during the period of gonadal differentiation (days 35 to 71 pf) resulted in phenological feminisation, i.e., all fish examined (n=28) showed an ovarian gonadal morphology. While gonadal CYP19A expression was suppressed compared to control fish, cerebral CYP19B (brain-derived CYP19 gene) mRNA expression was increased in 71-day-old MT-treated fish. The results from this study provide evidence that exogenous compounds interfering with the P450arom system are able to disrupt, even persistently, gonadal sex differentiation of the protogynic zebrafish. [Copyright &y& Elsevier]

Exposure of adult fathead minnows (Pimephales promelas) to the androgen 17α-methyltestosterone (MT) produces both androgenic and estrogenic effects, manifested as nuptial tubercle formation in females, and vitellogenin production in males and females, respectively. The present study was conducted to determine if the unanticipated estrogenic effects are produced by conversion of MT via aromatase activity to 17α-methylestradiol (ME2). Aromatase activity at the end of a 7-day waterborne MT exposure (20, 200 μg/l) was significantly decreased in ovarian microsomes and brain homogenates from exposed fish, to about 30–50% of control activity. Although aromatase activity was decreased by 7 days, it is possible that the conversion of MT to ME2 occurred soon after initial exposure. In support of this, ME2 was detected in plasma samples of the fish following the 7-day exposure, confirming their ability convert the androgen MT to the estrogen ME2. The concentration of ME2 in plasma was within the range of plasma 17ß-estradiol (E2) found in control female fathead minnows (4–5 ng/ml). These results, in conjunction with competitive binding assays that indicate ME2 binds to the fathead minnow estrogen receptor with a relative binding affinity of 68.3% of E2, support the hypothesis that aromatization of MT to ME2 contributes to the estrogenic effects in fathead minnows following exposure to this androgen. [Copyright &y& Elsevier]

Oestrogens are key regulators in sexual differentiation and development in higher vertebrates. P450 aromatase (P450arom) is the steroidogenic enzyme responsible for the synthesis of oestrogens from aromatisable androgens. Effects of endocrine disrupting chemicals on steroidogenic enzyme gene expression have received little attention so far, yet it is potentially a major pathway for sexual disruption. In this 14-day study the effects of exogenous 17β-oestradiol (E2) at environmentally relevant concentrations were assessed on gene expression of P450aromB in the gonad and brain of maturing male and female fathead minnows (FHM). Exposure to E2 resulted in an oestrogenic response as shown by a dose-dependent induction of plasma vitellogenin (VTG) in female and male fish and a dose-dependent inhibition of testis growth. There was an effect of exposure to E2 on P450aromB mRNA expression in the gonads; E2 up-regulated P450aromB mRNA expression in the testis and ovary in a dose-response manner after 14 days of exposure. In male brain, P450aromB mRNA concentrations were significantly reduced in fish exposed to 100 and 320 ng E2/l on day 4, but on day 14 were elevated in males exposed to both 32 and 100 ng E2/l. No effects of E2 on P450aromB mRNA expression occurred in the brain of females. The results of this study show that concentrations of E2 found in the environment can have disruptive effects on key steroidogenic enzyme pathways that control sexual development in fish. [Copyright &y& Elsevier]

• Aromatase inhibitors can cause reproductive dysfunction in group-synchronous fishes. • Fadrozole altered temporal changes in ovarian cyp19a , plasma E2, and hepatic vtg s. • Fadrozole caused cessation in ovarian recrudescence as demonstrated through low GSI. • Western mosquitofish are at least as sensitive to fadrozole as fathead minnow. Exposure to certain anthropogenic chemicals can inhibit the activity to cytochrome P450 aromatase (CYP19) in fishes leading to decreased plasma 17β-estradiol (E2), plasma vitellogenin (VTG), and egg production. Reproductive dysfunction resulting from exposure to aromatase inhibitors has been extensively investigated in several laboratory model species of fish. These model species have ovaries that undergo asynchronous oocyte development, but many fishes have ovaries with group-synchronous oocyte development. Fishes with group-synchronous oocyte development have dynamic reproductive cycles which typically occur annually and are often triggered by complex environmental cues. This has resulted in a lack of test data and uncertainty regarding sensitivities to and adverse effects of aromatase inhibition. The present study used the western mosquitofish (Gambusia affinis) as a laboratory model to investigate adverse effects of chemical aromatase inhibition on group-synchronous oocyte development. Adult female western mosquitofish were exposed to either 0, 2, or 30 μg/L of the model nonsteroidal aromatase inhibiting chemical, fadrozole, for a complete reproductive cycle. Fish were sampled at four time-points representing pre-vitellogenic resting, early vitellogenesis, late vitellogenesis/early ovarian recrudescence, and late ovarian recrudescence. Temporal changes in numerous reproductive parameters were measured, including gonadosomatic index (GSI), plasma sex steroids, and expression of selected genes in the brain, liver, and gonad that are important for reproduction. In contrast to fish from the control treatment, fish exposed to 2 and 30 μg/L of fadrozole had persistent elevated expression of cyp19 in the ovary, depressed expression of vtg in the liver, and a low GSI. These responses suggest that completion of a group-synchronous reproductive cycle was unsuccessful during the assay in fish from either fadrozole treatment. These adverse effects data show that exposure to aromatase inhibitors has the potential to cause reproductive dysfunction in a wide range of fishes with both asynchronous and group-synchronous reproductive strategies. [ABSTRACT FROM AUTHOR]

• cyp19a1a-eGFP zebrafish exposed to PCZ show similar responses to wild type. • The cyp19a1a-eGFP line allows in vivo monitoring of gonad fluorescence over time. • The cyp19a1a-eGFP provides additional mechanistic information to the TG229/230 test. Transgenic fish are powerful models that can provide mechanistic information regarding the endocrine activity of test chemicals. In this study, our objective was to use a newly developed transgenic zebrafish line expressing eGFP under the control of the cyp19a1a promoter in the OECD Fish Short Term Reproduction Assay (TG 229) to provide additional mechanistic information on tested substances. For this purpose, we exposed adult transgenic zebrafish to a reference substance of the TG 229, i.e. prochloraz (PCZ; 1.7, 17.2 and 172.6 μg/L). In addition to "classical" endpoints used in the TG 229 (reproductive outputs, vitellogenin), the fluorescence intensity of the ovaries was monitored at 4 different times of exposure using in vivo imaging. Our data revealed that 172.6 μg/L PCZ significantly decreased the number of eggs laid per female per day and the concentrations of vitellogenin in females, reflecting the decreasing E2 synthesis due to the inhibition of the ovarian aromatase activities. At 7 and 14 days, GFP intensities in ovaries were similar over the treatment groups but significantly increased after 21 days at 17.2 and 172.6 μg/L. A similar profile was observed for the endogenous cyp19a1a expression measured by qPCR thereby confirming the reliability of the GFP measurement for assessing aromatase gene expression. The overexpression of the cyp19a1a gene likely reflects a compensatory response to the inhibitory action of PCZ on aromatase enzymatic activities. Overall, this study illustrates the feasibility of using the cyp19a1a - eGFP transgenic line for assessing the effect of PCZ in an OECD test guideline while providing complementary information on the time- and concentration-dependent effects of the compound, without disturbing reproduction of fish. The acquisition of this additional mechanistic information on a key target gene through in vivo fluorescence imaging of the ovaries was realized without increasing the number of individuals. [ABSTRACT FROM AUTHOR]

Abstract: Significant amounts of oil and alkylphenols are released into the sea by petroleum installations as a result of discharges of produced water. Some of these pollutants elicit estrogenic responses in fish, but their effects on the endocrine system of molluscs are largely unknown. In this study, mussels Mytilus edulis were exposed to North Sea oil (O) and the mixture of North Sea oil+alkylphenols (OAP), and the effects on tissue steroid levels and steroid metabolism (P450-aromatase and estradiol-sulfotransferase) were monitored. Levels of free testosterone and free estradiol were much higher in gonad tissue than in peripheral tissue, whereas esterified steroids (released after saponification) were of the same order of magnitude in both tissues. Levels of free steroids determined in gonads were not affected by exposure, but esterified steroids significantly increased in OAP exposed mussels (up to 2.4-fold). The sulfation of estradiol was investigated as a conjugation pathway, and increased activities were observed in digestive gland cytosol of both O and OAP exposure groups (up to 2.8-fold). Additionally, increased P450-aromatase activity was determined in OAP exposed mussels (up to three-fold, both in gonad and digestive gland), but not in the O group. Altogether, the results indicate that North Sea oil leads to increased sulfation of estradiol, and that in combination with alkylphenols, additional alterations are observed: increased P450-aromatase, and increased levels of esterified-steroids in gonads. Nonetheless, mussels are able to maintain gonad concentrations of free steroids unaltered, possibly via homeostatic mechanisms such as the conjugation with fatty acid or the formation of sulphate conjugates. [Copyright &y& Elsevier]

Abstract: Juvenile Atlantic cod (Gadus morhua) and turbot (Scophthalmus maximus) were exposed for 3 weeks in a continuous water flow to 0.5ppm of dispersed North Sea crude oil, 0.5ppm of dispersed North Sea crude oil spiked with 0.1ppm of a mixture of alkylphenols (offshore oil production), and 30ppb of nonylphenol (NP). As potential markers of endocrine alteration, key enzymatic activities involved in both synthesis (17β-hydroxysteroid dehydrogenases and P450 aromatase) and metabolism (liver UDP-glucuronosyltransferases (UGT) and sulfotransferases) of steroids were assessed together with circulating levels of testosterone and estradiol in plasma. NP-exposed turbot had lower ovarian P450 aromatase, lower levels of testosterone and estradiol in plasma, and lower glucuronidation rates of sex steroids than those from the control group. In contrast, higher liver UGT-testosterone, and a trend towards higher P450 aromatase was detected in oil-exposed specimens. Those exposed to the combination oil+alkylphenols had lower levels of estradiol in plasma than controls, and no significant effects on any the enzymatic activities tested was observed. All these alterations were more evident in turbot than in cod. In fact, apart from a higher glucuronidation rate of estradiol detected in the liver of NP-exposed cod, no significant differences were observed between control and exposed cod. [Copyright &y& Elsevier]