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In the present study, we investigated the dopaminergic and steroid hormone systems of A/J mice fed environmentally relevant concentrations of a perfluoroalkyl substance (PFAS) mixture over a period of 10 weeks. The PFAS mixture was chosen based on measured PFAS concentrations in earthworms at a Norwegian skiing area (Trondheim) and consisted of eight different PFAS. Dietary exposure to PFAS led to lower total brain dopamine (DA) concentrations in male mice, as compared to control. On the transcript level, brain tyrosine hydroxylase (th) of PFAS exposed males was reduced, compared to the control group. No significant differences were observed on the transcript levels of enzymes responsible for DA metabolism, namely – monoamine oxidase (maoa and maob) and catechol-O methyltransferase (comt). We detected increased transcript level for DA receptor 2 (dr2) in PFAS exposed females, while expression of DA receptor 1 (dr1), DA transporter (dat) and vesicular monoamine transporter (vmat) were not affected by PFAS exposure. Regarding the steroid hormones, plasma and muscle testosterone (T), 11-ketotestosterone (11-KT) and 17β-estradiol (E2) levels, as well as transcripts for estrogen receptors (esr1 and esr2), gonadotropin releasing hormone (gnrh) and aromatase (cyp19) were unaltered by the PFAS treatment. These results indicate that exposure to PFAS doses, comparable to previous observation in earthworms at a Norwegian skiing area, may alter the dopaminergic system of mice with overt consequences for health, general physiology, cognitive behavior, reproduction and metabolism.

Perfluoroalkyl substances (PFAS) are contaminants that are applied in a wide range of consumer products, including ski products. The present study investigated the neuro-dopamine (DA) and cellular steroid hormone homeostasis of wild Bank voles (Myodes glareolus) from a skiing area in Norway (Trondheim), in relation to tissue concentrations of PFAS. We found a positive association between brain DA concentrations and the concentration of several PFAS, while there was a negative association between PFAS and dopamine receptor 1 (dr1) mRNA. The ratio between DA and its metabolites (3,4-dihydroxyphenylacetic acid: DOPAC and homovanillic acid: HVA) showed a negative association between DOPAC/DA and several PFAS, suggesting that PFAS altered the metabolism of DA via monoamine oxidase (Mao). This assumption is supported by an observed negative association between mao mRNA and PFAS. Previous studies have shown that DA homeostasis can indirectly regulate cellular estrogen (E2) and testosterone (T) biosynthesis. We found no association between DA and steroid hormone levels, while there was a negative association between some PFAS and T concentrations, suggesting that PFAS might affect T through other mechanisms. The results from the current study indicate that PFAS may alter neuro-DA and steroid hormone homeostasis in Bank voles, with potential consequences on reproduction and general health. This is an open access article distributed under the terms of the Creative Commons CC-BY license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Receptor (i.e. genomic) and non-receptor (or non-genomic) effects of endocrine toxicology have received limited or almost non-existent attention for tropical species and regions. In the present study, we have evaluated the effects of di-(2-ethylhexyl) phthalate (DEHP) on neuro- and interrenal steroidogenesis of the African catfish (Clarias gariepinus) using molecular, immunochemical and physiological approaches. Juvenile fish (mean weight and length: 5.6±0.6g and 8.2±1.2cm, respectively), were randomly distributed into ten 120L rectangular glass tanks containing 60L of dechlorinated tap water, at 50 fish per exposure group. The fish were exposed to environmentally relevant concentrations of DEHP, consisting of 0 (ethanol solvent control), 10, 100, 200, and 400μg DEHP/L water and performed in two replicates. Brain, liver and head kidney samples were collected at day 3, 7 and 14 after exposure, and analysed for star, p450scc, cyp19a1, cyp17, cyp11β-, 3β-, 17β- and 20β-hsd, and 17β-ohase mRNA expression using real-time PCR. The StAR, P450scc and CYP19 proteins were measured using immunoblotting method, while estradiol-17β (E2) and testosterone (T) were measured in liver homogenate using enzyme immunoassay (EIA). Our data showed a consistent and unique pattern of biphasic effect on star and steroidogenic enzyme genes with increases at low concentration (10μg/L) and thereafter, a concentration-dependent decrease in both the brain and head kidney, that paralleled the expression of StAR, P450scc and CYP19 proteins. Cellular E2 and T levels showed an apparent DEHP concentration-dependent increase at day 14 of exposure. The observed consistency in the current findings and in view of previous reports on contaminants-induced alterations in neuro- and interrenal steroidogenesis, the broader toxicological and endocrine disruptor implication of our data indicate potentials for overt reproductive, metabolic, physiological and general health consequences for the exposed organisms.

In the present study, we investigated the occurrence of intersex condition, histopathological changes in the gonad and endocrine disruptor biomarker responses in Tilapia species (Tilaipia guineensis, Sarotherodon galileaus and Oreochromis niloticus) along the Ogun River, Nigeria. The study sites covered a length of 320 km and a total of 1074 tilapias were collected from three sampling sites (Abeokuta, Isheri and Ikorodu) with different degrees of anthropogenic contamination. Samples were also collected from an upstream putative control site (Igboho) along the Ogun River. Hepatic transcript levels for vitellogenin (Vtg), zona radiata (Zrp) and aromatase (cyp19a1) were analyzed using real-time PCR. Gross gonadal morphology revealed a 24% prevalence of intersex showing visible testis and ovary in phenotypic females (25.4%) or males (74.6%). The intersex condition paralleled histopathological changes (ovotestis or testis-ova) in the gonads of female and male fish, respectively. Plasma concentrations of luteinizing hormone (LH), follicle stimulating hormone (FSH), 11-ketotestosterone (11-KT) and estradiol-17β (E2) were measured using enzyme immunoassay, showing that male fish from downstream of the control site had significantly higher plasma E2, LH, and FSH concentrations compared to females. Similarly, Vtg, Zrp and cyp19a1 mRNA was significantly higher in males, compared to females. Analysis of contaminants showed the presence of 15 PCB congeners, lindane and dieldrin, and 4-iso-nonylphenol (4-iso-NP) and 4-tert-octylphenol (4-tert-OP) in fish muscle and sediment samples from Ogun River. Principal component analysis (PCA) revealed site and sex relationships between measured biological responses to groups of environmental contaminants, showing that the endocrine disruptive responses in fish were associated with biota and sediment contaminant burden. In addition, strong positive correlations were observed between male fish and Zrp, cyp19a1, E2, LH, FSH, PCBs, 4-iso-NP and 4-tert-OP, suggesting possible feminization effects of these contaminants on the male. In female fish, PCBs, 4-iso-NP and 4-tert-OP showed positive relationships with 11-KT and gonadosomatic index (GSI), suggesting masculinization effects by these contaminants. Overall, our findings demonstrate a causal relationship between endocrine disruption and contaminants burden in Tilapias species from Ogun River.

In the present study, we investigated plasma biochemical and steroid hormone responses, together with gonado-histopathological alterations in Clarias gariepinus exposed to sublethal concentrations of two synthetic pyrethroids (cypermethrin and deltamethrin). Fish were exposed to environmentally-relevant concentrations of cypermethrin at 0 (ethanol solvent control), 0.07, 0.014, 0.028, 0.056) and deltamethrin at 0.22, 0.44, 0.88 and 1.76 μg/L, for 7, 14, 21 and 28 days. Plasma enzyme (aspartate transaminase: AST, alanine transaminase: ALT and alkaline phosphatase: ALP) and steroid hormones (estradiol-17β: E2, testosterone: T) levels were analyzed. Gonado-histopathological evaluation shows the presence of ovo-testis (intersex), oocytes atresia, cytoplasmic degeneration and clumping of vitellogenic oocytes in females, while male fish displayed enlargement and degeneration of testicular seminiferous tubules after 28 days exposure to cypermethrin and deltamethrin. Plasma biochemical analysis in pesticides exposed fish revealed that AST, ALT and ALP were significantly increased in a concentration-dependent manner. In addition, we observed respective and apparent concentration- and time-dependent increase and decrease of plasma E2 and T levels, compared to control. Interestingly, the significant increase in E2 levels paralleled gonadal ovo-testis (intersex) condition in exposed fish, indicating endocrine disruptive effects of cypermethrin and deltamethrin that favor the estrogenic pathway, in addition to overt negative consequences on reproductive, biochemical and physiological health of the exposed fish.

The dopaminergic effect of PAH and PFAS mixtures, prepared according to environmentally relevant concentrations, has been studied in juvenile female Atlantic cod (Gadus morhua). Benzo[a]pyrene, dibenzothiophene, fluorene, naphthalene, phenanthrene, and pyrene were used to prepare a PAH mixture, while PFNA, PFOA, PFOS, and PFTrA were used to prepare a PFAS mixture. Cod were injected intraperitoneally twice, with either a low (1×) or high (20×) dose of each compound mixture or their combinations. After 2 weeks of exposure, levels of plasma 17β-estradiol (E2) were significantly elevated in high PAH/high PFAS treated group. Brain dopamine/metabolite ratios (DOPAC/dopamine and HVA+DOPAC/dopamine) changed with E2 plasma levels, except for high PAH/low PFAS and low PAH/high PFAS treated groups. On the transcript levels, th mRNA inversely correlated with dopamine/metabolite ratios and gnrh2 mRNA levels. Respective decreases and increases of drd1 and drd2a after exposure to the high PAH dose were observed. Specifically, high PFAS exposure decreased both drds, leading to high plasma E2 concentrations. Other studied end points suggest that these compounds, at different doses and combinations, have different toxicity threshold and modes of action. These effects indicate potential alterations in the feedback signaling processes within the dopaminergic pathway by these contaminant mixtures. acceptedVersion

In the present study, the developmental (including fertility) and endocrine-disruptive effects in relation to chemical burden in male and female Nile crocodiles (Crocodylus niloticus), from a commercial crocodile farm in the Brits district, South Africa, exposed to various anthropogenic aquatic contaminants from the natural environment was investigated. Hepatic transcript levels for vitellogenin (Vtg), zona pellucida (ZP) and ERα (also in gonads) were analyzed using real-time PCR. Plasma estradiol-17β (E2), testosterone (T) and 11-ketotestosterone (11-KT) were analyzed using enzyme immunoassay. Gonadal aromatase and hepatic testosterone metabolism (6β-hydroxylase (6β-OHase)) were analyzed using biochemical methods. Overall, there is high and abnormal number (%) of infertile and banded eggs during the studied reproductive seasons, showing up to 57 and 34% of infertile eggs in the 2009/2010 and 2013/2014 seasons, respectively. In addition, the percentage of banded eggs ranged between 10 and 19% during the period of 2009-2014 seasons. While hepatic ERα, Vtg, ZP mRNA and testosterone 6β-OHase, were equally expressed in female and male crocodiles, gonadal ERα mRNA and aromatase activity were significantly higher in females compared to male crocodiles. On the other hand, plasma T and 11-KT levels were significantly higher in males, compared to female crocodiles. Principal component analysis (PCA) produced significant grouping that revealed correlative relationships between reproductive/endocrine-disruptive variables and liver contaminant burden, that further relates to measured contaminants in the natural environment. The overall results suggest that these captive pre-slaughter farm crocodiles exhibited responses to anthropogenic aquatic contaminants with potentially relevant consequences on key reproductive and endocrine pathways and these responses may be established as relevant species endocrine disruptor biomarkers of exposure and effects in this threatened species.

Aiming to explore ultrasound technology as a noninvasive method for maturation monitoring, we compared ultrasound observations and measurements in female Atlantic salmon (Salmo salar) during the last year before ovulation with standard, invasive methods such as gonadosomatic index (GSI), gonad histology and sex hormone analysis. Ultrasound measurements of ovaries correlated strongly (R > 0.9, P

In the present study, we have investigated the effects of 30-day dietary (pre-pubertal) exposure to different doses (0 (control), 1, 10, 50, 200 and 400 mg/kg bodyweight/day) of di(n-butyl) phthalate (DBP) on Leydig cells of adult male Japanese quails by quantifying the transcript levels for P450 side-chain cleavage (p450scc), P450c17 (CYP17), and 3β- and 17β-hydroxysteroid dehydrogenase (hsd) using quantitative (real-time) polymerase chain reaction (qRT-PCR). In addition, the plasma testosterone levels were analysed using radioimmunoassay (RIA) and testis was examined for evidence of gross pathology and histopathology. Our data showed that pre-pubertal exposure to DBP produced alterations in testicular architecture as evident by poorly developed or mis-shaped testis, and altered spermatogenesis due to tubular degeneration and atrophy of seminiferous tubules especially in the high DBP dose (200 and 400 mg/kg) treated groups. In addition, DBP altered several key enzymes involved in testicular steroidogenesis pathways in an apparent dose-dependent manner. For example, biphasic effects of DBP were observed for P450scc and 3β-hsd mRNA, that were generally increasing at low dose 10 mg/kg, and thereafter, an apparent dose-dependent decrease between 50 and 400mg/kg. The steroidogenic acute regulatory (StAR) protein was at the lowest detectable limits and therefore not quantifiable. These effects did not parallel the non-significant changes observed for plasma testosterone levels. The present data is consistent with previous reports showing that DBP modulates Leydig cell steroidogenesis in several species, with a potential negative effect on reproduction in those avian species that are vulnerable to endocrine disrupting chemicals.

In the present study, we have used salmon embryos whose continuous exposure to waterborne PFOA or PFOS at 100 μg/L started as freshly fertilized eggs, and lasted for a total of 52 days. PFOS and PFOA were dissolved in methanol (carrier vehicle) whose concentration never exceeded 0.01% of total tank volume. Samples were collected at day 21, 28, 35, 52, 49 and 56 after the start of the exposure. Note that days 49 and 56 represent end of exposure and 1 week after a recovery period, respectively. Tissue bioaccumulations were determined by HPLC/MS/MS, steroid hormones, fatty acids (FAs) and lipids were determined by GC–MS, while mRNA expression levels of genes were determined by qPCR in whole body homogenate. We observed that PFOS and PFOA showed a steady increase in whole body burden during the exposure period, with a slight decrease after the recovery period. Calculated somatic indexes showed that PFOA produced increases in heart-, thymus-, liver- and kidney somatic indexes (HSI, TSI, LSI and KSI). PFOA and PFOS exposure produced significant decreases in whole body dehydroepiandrosterone (DHEA), estrone and testosterone at sampling day 21 and a strong increase of cortisol and cholesterol at the end of recovery period (day 56). PFOA and PFOS effects differed with DHEA and estrone. While PFOS decreased DHEA levels, PFOA produced an increase at day 49, and while PFOS decreased estrone, PFOA produced a slight increase at day 56. We observed changes in FA composition that predominantly involved increases in FA methyl esters (FAMEs), mono- and poly-unsaturated FA (MUFA and PUFA) and a decrease in n-3/n-6 PUFA ratio by both PFOA and PFOS. Particularly, an increase in – pentadecenoic MUFA (15:1), two n-3 PUFAs α-linolenic acid [ALA: 18:3 n3] and eicosapentaenoic acid [EPA: 20:5 n-3] and n-6 PUFA: arachidonic acid [ARA: 20:4 n6], docosapentaenoic acid (DPA) by PFOA and PFOS were observed. These effects were associated with changes in mRNA expression of FA elongase (FAE), Δ5-desaturase (FAD5) and Δ6-desaturase (FAD6) genes. In summary, the changes in hormonal and FA profiles may represent cellular and/or physiological adaptation to continuous PFOS and PFOA exposure by increasing membrane fluidity, and/or overt developmental effects. The present findings provide some potential insights and basis for a better understanding on the possible mechanisms of PFCs toxicity in fish.

The present study was conducted to evaluate the effects of perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA) on lipid β-oxidation and oxidative stress responses in Atlantic salmon liver and kidney tissues. We quantified changes in the expression levels of peroxisome proliferator-activated receptors (PPARs) and acyl-CoA oxidase (ACOX1) enzyme whose transcription is induced by PPARs. In addition, we analyzed gene expression patterns for enzymatic antioxidants (superoxide dismutase: SOD, catalase: CAT and glutathione peroxidase: GPx). Thiobarbituric acid reactive substances (TBARS) were analyzed as a measure for lipid peroxidation. Juvenile Atlantic salmon were repeatedly force-fed food spiked with PFOA or PFOS at 0.2mg/kg, and samples were collected after 0, 2, 5 and 8 days and after a 7 days recovery period. Our data showed that exposure of salmon to PFOS or PFOA produced changes (either increased or decreased) in mRNA expression for PPARs, ACOX1, oxidative stress responses and lipid peroxidation (TBARS) and these responses showed marked organ differences, associated with tissue bioaccumulation patterns and dependent on exposure time. Given that a classical reaction during reactive oxygen species (ROS)-induced damage involves the peroxidation of lipids, our study demonstrates that salmon continuously exposed to dietary PFOS or PFOA dose showed alteration in peroxisomal responses and oxidative stress responses, with higher severity in the kidney, compared to liver. Overall, our data suggest that ROS-mediated oxidative damage maybe a significant and putative toxic effect of PFOA and PFOS in fish as has been reported in mammals.

Predictive and reliable parameters of reproductive status are integral aspects of sustainable fisheries and aquaculture management. These parameters are also important for an accurate evaluation of the effects of different treatments on sexual maturation in fish farming. In the present study, we have characterized the seasonal reproduction profile and described changes in sex steroids in relation to gonadal maturation and development in female and male Waigieu seaperch (Psammoperca waigiensis). The experimental period covered a full calendar year (January–December). In males and females, we observed that plasma sex steroid hormones [oestradiol-17β (E2), testosterone (T), 11-ketotestosterone (11-KT) and progesterone (P)] levels showed monthly fluctuations during the spawning period. Particularly, plasma steroid hormone levels were positively associated with gonadosomatic index values. In addition, high levels of plasma steroid coincided with recruitment of oocytes into yolk accumulation in females. The main spawning period occurred between April and October in females, and between March and November in males. The non-aromatizable androgen, 11-KT is generally believed to be the active male-specific androgen in teleosts, and is associated with the process of spermiation, development of secondary sexual characteristics and regulation of male reproductive behaviour in most teleost species. In this study, we found relatively high amounts of 11-KT in females between May and December, suggesting an integral role in the maturation process, also for the females. A rapid peak in plasma P level was observed in November and suggests significant roles during post-spawning and/or resting periods in both female and male fish. Furthermore, all oocyte developmental stages were present within the same sampling month and also within the spawning period, demonstrating the gamete group asynchronous developmental strategy. Overall, Waigieu seaperch showed strong seasonality in reproductive development with corresponding sex steroid patterns. The data presented in this study may contribute to the understanding of the reproductive endocrinology of a tropical marine finfish with increasing industrial prospects and sustainable aquaculture of this species in a developing country, such as Vietnam.

Tributyltin (TBT) is a widespread marine pollutant that influences physiological conditions of fish and other aquatic organisms. In addition to effects on reproduction, the immune system has been proposed as a possible target for TBT effects. In the present study, the effects of TBT exposure were examined on the expression of genes involved in immune system compentence in liver and head kidney of Atlantic salmon, in the presence and absence of a second-messenger activator (forskolin). Juvenile salmon were force-fed a diet containing TBT (0-solvent control, 0.1, 1, or 10 mg/kg fish) for 72 h. Consequently, fish from the control group and 10-mg/kg TBT group were exposed to the adenylate cyclase (AC) activator forskolin (200 μg/L) for 2 or 4 h. Forskolin was selected for this study because it is known to exhibit potent immune system enhancement by activating macrophages and lymphocytes. After sacrifice, liver and head kidney were sampled and transcript changes for interleukin (IL)-1β, IL-10, transforming growth factor (TGF) β, interferon (INF) α, INFγ, tumor necrosis factor (TNF) α, Mx3, and insulin-like growth factor (IGF)-1 were determined in both tissues by quantitative polymerase chain reaction (qPCR) using gene-specific primers. TBT, when given alone and also in combination with forskolin, decreased IL-1β, TNFα, IFNγ, IFNα, Mx3, and IGF-1 gene expression. In contrast, IL-10 and TGFβ transcripts were increased after TBT exposure alone and also in combination with forskolin. Generally, these effects were largely dependent on TBT dose and time of exposure when given in combination with forskolin. Overall, our findings suggest a possible immunomodulatory effect of TBT, possibly involving cAMP activation.

Environmentally relevant concentrations of selected polychlorinated biphenyl (PCB) and polybrominated diphenyl ether (PBDE) flame retardant congeners and their hydroxylated (OH) and methoxylated (MeO) analogues that can perturb thyroid hormone-dependent processes were comparatively examined with respect to competitive binding with thyroxine (T(4)) and 3,5,3'-triiodothyronine (T(3)) thyroid hormones (THs) on recombinant human and gull albumin and transthyretin transport proteins. The liver tissue was from glaucous gulls (Larus hyperboreus) from Norway and herring gulls (Larus argentatus) from the Great Lakes of North America. We isolated, cloned, sequenced, purified, and expressed the cDNA (cDNA) of albumin from liver of herring and glaucous gull. Albumin amino acid sequences were identical for both gull species. Concentration-dependent, competitive binding curves were generated for T(4) and T(3) binding alone and for selected substrates using gull and human recombinant albumin (recALB). Human recALB had high preference for T(4) relative to T(3), whereas it was reversed for gull recALB. Binding assays with recALB and recTTR gull proteins showed that relative to 2,2',4,4'-tetrabromoDE (BDE-47) and 2,2',3,4',5,5',6-heptaCB (CB-187) and the MeO-substituted (4-MeO-CB187 and 6-MeO-BDE47) analogues, 4-OH-CB187, 6-OH-BDE47, and 4'-OH-BDE49 had the greatest binding affinity and potency, and that competitive binding was greater for T(3) relative to T(4). These results indicate that xenobiotic ligand binding to human ALB or TTR cannot be used as a surrogate for gull binding interactions. The combination of TH-like brominated diphenyl ether backbone (relative to the chlorinated biphenyl backbone), and the presence of OH-group produced a more effective competitive ligand on human and gull recALB and recTTR relative to both T(3) and T(4). This suggests the possibility that OH-substituted organohalogen contaminants may be an exposure concern to the thyroid system in free-ranging gulls as well as for humans.

At present, there are no known direct occurrences of nonylphenol (NP) in nature. Therefore, its presence in nature is solely a consequence of human activities. NP is generated through degradation of alkylphenol ethoxylates released mainly from textile, metal working, institutional cleansing and laundry cleaning, but few data on the amount of the release is available. These compounds have been shown to affect several biological processes, including the endocrine systems, in a wide number of species. The cytochrome P450 aromatase (Cyp19) is the rate-limiting step in estrogen production, and is known to be a potential target for endocrine-disrupting chemicals (EDCs) such as NP. Teleost fish generally have a high brain aromatase activity, and the effects of EDCs in fish brain is not thoroughly investigated. In this study, juvenile Atlantic salmon (Salmo salar) were exposed to waterborne concentrations of the synthetic pharmaceutical and xenoestrogen 17alpha-ethynylestradiol (EE2; 5ng/L) and the xenoestrogen 4-nonylphenol (NP; 5 and 50microg/L) for 72h. Brain tissue and blood were sampled from individual fish. Gene expression patterns of Cyp19 isoforms were determined by quantitative PCR, aromatase protein immunoreactivity in the brain was evaluated by immunohistochemistry and immunoblotting, and aromatase activity was analyzed using the tritiated water-release assay. Plasma estradiol (E2) and testosterone (T) levels were measured by EIA. In the brain, EE2 increased the mRNA expression of Cyp19b almost threefold compared to the solvent control, whereas Cyp19a levels were unaffected by EE2 treatment. In contrast, both NP concentrations produced significant reduction of Cyp19a expression. Immunohistochemical aromatase protein reactivity was localized in several brain regions, but no apparent quantitative effects of the exposures were observed. Immunoblotting analysis showed that EE2 and NP produced a slight increase in brain immunoreactive aromatase protein band, compared with controls. Plasma levels of E2 increased twofold when treated with EE2 and 5microg NP/L, and threefold when exposed to 50microg NP/L. In general, the present study shows that the parallel biochemical, transcriptional and cellular detection of neural aromatase for endocrine-disrupting effects from EE2 and NP may be observed at specific levels of the biological organization.

Available evidence suggests that androgens play critical roles in early oocyte growth and development in fish. However, the molecular mechanisms underlying this important aspect of reproductive endocrinology have not yet been established. In this study the effects of androgens (11-ketotestosterone [11-KT] and testosterone [T]) were determined on gene expression patterns and growth of cod previtellogenic oocytes, using an in vitro oocyte culture technique. Previtellogenic ovarian tissue was cultured for 5 and 10 d at different concentrations of 11-KT and T (0, 1, or 1000 microM) dissolved in ethanol (0.3%). The androgen concentrations were selected as they represent physiological and supra-physiological concentrations, respectively. Quantitative polymerase chain reaction (PCR) demonstrated increased mRNA expression for five genes recently identified as androgen responsive in our subtracted cDNA library in previtellogenic cod ovary exposed in vitro to androgens. Quantitative histological analyses showed a consistent stereological validation of oocyte growth and development after exposure to androgens. In general, both 11-KT and T induced previtellogenic oocyte growth and development, and these effects were more pronounced with 11-KT exposure. Taken together, our study reveals some novel roles of androgens on the development of previtellogenic oocytes, indicating control of early follicular and oocyte growth in cod ovary. The potent effects of 11-KT on oocyte growth support our earlier hypothesis that non-aromatizable androgens play significant roles in regulating early oocyte growth with potential consequences for the fecundity process. Therefore, these novel roles of androgens as promoters of ovarian growth and development presented in this study may be useful for the aquaculture industry and for breeding of new captive and endangered species. From a toxicological point of view, the cod is a marine species and exposure to complex chemical mixtures that may exert androgenic and/or anti-androgenic effects represents an environmental issue of reasonable concern in the marine environment. Therefore, the findings in the present study represent a novel basis that can be used to determine the effects of xenoandrogens on oocyte development and fecundity in this important marine species.

The induction of CYP enzyme activities, particularly CYP1A1, through the aryl hydrocarbon receptor (AhR) in most vertebrate species is among the most studied biochemical response to planar and aromatic organic contaminant exposure. Since P450 families play central roles in the oxidative metabolism of a wide range of exogenous and endogenous compounds, interactions between the biotransformation processes and reproductive physiological responses are inevitable. Steroidogenesis is the process by which specialized cells in specific tissues, such as the gonad, brain (neurosteroids) and kidney, synthesize steroid hormones. In the present study, we evaluated the effects of water-soluble fraction (WSF) of crude oil on the xenobiotic biotransformation and steroidogenic processes in the head (brain) and whole-body tissue of a model species by transcript analysis using quantitative (real-time) polymerase chain reaction (qPCR), enzyme activities and steroid hormone (testosterone: T and 17beta-estradiol: E2) levels using enzyme immune assay (EIA). Our data showed that exposure of fish to WSF produced an apparent concentration-specific increase of AhR1, CYP1A1 and 3beta-hydroxysteroid dehydrogenase (3beta-HSD) mRNA levels, and decrease of AhR2. On the activity level, WSF produced concentration-specific increase of ethoxyresorufin O-deethylase (EROD), benzyloxyresorufin (BROD) methoxyresorufin (MROD) and pentoxyresorufin (PROD) activities in whole-body tissue. In the steroidogenic pathway, WSF exposure produced apparent concentration-specific decrease of ER* and ERbeta, steroidogenic acute regulatory (StAR) protein, cytochrome P450 side-chain cleavage (P450scc), P450aromA and P450aromB mRNA expression. For steroid hormones, while T levels decreased, E2 levels increased in an apparent WSF concentration-specific manner. In general, the xenobiotic biotransformation and estrogenic responses showed negative relationship after exposure of zebrafish to WSF, suggesting an interaction between these physiological pathways. The relationship between WSF mediated changes in brain StAR, P450scc, 3beta-HSD, ER*alpha, ERbeta, P450aromA, P450aromB and whole-body steroid hormone levels suggests that the experimental animals might be experiencing altered neurosteroidogenesis probably through increased activity level of the biotransformation system. Thus, these responses might represent sensitive diagnostic tools for short-term and acute exposure of fish or other aquatic organisms to WSF.

The aim of the present study is to investigate the endocrine disrupting potential of the explosive 2,4,6-trinitrotoluene (TNT) on marine organisms using the European eel (Anguilla anguilla Linnaeus, 1758) as model fish species. Crucial brain steroidogenic parameters such as the estrogen receptors (ERs), P450 aromatase (CYP19), steroidogenic acute regulatory (StAR) protein and P450-mediated cholesterol side-chain cleavage (P450scc) enzyme were selected as potential biomarkers for the assessment of TNT endocrine disrupting potential. Juvenile European eels were exposed for 6 and 24 h to 0.5, 1 and 2.5 mg/l nominal concentration of TNT dissolved in dimethyl sulfoxide (DMSO). ERα, CYP19, StAR protein and P450scc genes expression were quantified using real-time PCR. Exposure to TNT altered the expression of ER gene at the lowest TNT concentration after 6 h of exposure, while after 24 h gene expression levels increased respect to controls only in eels at the maximum TNT dose (2.5 mg/l). A significant increase o...

Active derivatives of vitamin A are essential in physiological processes such as cell growth, differentiation, morphogenesis and development. The biological functions of vitamin A are mediated through the retinoid acid receptors (RARs) and retinoid X receptors (RXRs). Aryl hydrocarbon receptor (AhR) agonists such as planar halogenated compounds are known to interfere with vitamin A homeostasis in both field and laboratory studies. In this study, we have investigated the molecular interactions between vitamin A and AhR signalling pathways using juvenile Atlantic salmon and agonists for both receptor pathways. Groups of juvenile salmon were treated with all-trans- and 9-cis-retinoic acid mixture (7:3 ratio) dissolved in DMSO (dimethyl sulfoxide) at 0.1, 1 and 10 mg/kg fish weight. The mixture was force fed singly or in combination with 0.1 mg 3,3',4,4'-tetrachlorobiphenyl (co-planar congener 77)/kg fish weight dissolved in DMSO. Liver samples were collected 3 days after PCB-77 exposure. A separate group exposed to combined retinoic acid (1 mg/kg for 5 days) and PCB-77, was sampled at 3, 7 and 14 days after PCB-77 exposure. Liver samples collected from all exposure groups were analyzed for gene (RARalpha, AhR2alpha, AhR2beta, CYP1A1, UGT1 and GSTpi) expression using real-time PCR and activity (7-ethoxyresorufin O-deethylase (EROD), UGT and GST) using biochemical methods with specific substrates. Our data showed that exposure to RA alone did not produce a significant increase of RARalpha mRNA levels, and the presence of PCB-77 attenuated the expression of RARalpha in RA dose- and time-specific manner. In addition, RA produced a dose-dependent increase of CYP1A1 mRNA and activity (EROD) levels without concomitant increase in AhR2 isoforms. When administered alone, PCB-77 produced increased CYP1A1, UGT1 and GSTpi mRNA and enzyme levels. The PCB-77-induced CYP1A1, UGT1 and GSTpi (mRNA and activity) levels were modulated by RA, in a parameter and dose-specific manner. In general, our data show an interaction between vitamin A and AhR signalling that may affect retinoid homeostasis in fish.

Gonadal steroids are known to modulate both the synthesis and the release of gonadotropins by the pituitary and influence several brain functions that are apparently responsible for gender-specific differences in the regulation of the hypothalamus-pituitary-gonadal (HPG) axis. It is believed that the true rate-limiting step in acute steroid production is the movement of cholesterol across the mitochondrial membrane by the steroidogenic acute regulatory (StAR) protein and subsequent conversion to pregnenolone by P450-mediated cholesterol side chain cleavage (P450 scc). In the present study, we have evaluated the effects of 17alpha-ethynylestradiol (EE2) on salmon previtellogenic oocytes using an in vitro culture system and molecular, histological, and physiological methods. The in vitro culture technique was based on an agarose floating method recently validated for xenoestrogens in our laboratory. Tissue was cultured in a humidified incubator at 10 degrees C for 3, 7, and 14 days with different concentrations of EE2 [0 (control), 0.01, 0.1, and 1 microM] dissolved in ethanol (0.1%). The StAR, P450 scc, P450 arom isoforms, and insulin-like growth factor 2 (IGF-2) mRNA expressions were performed using validated real-time polymerase chain reaction (PCR) with specific primers, and immunohistochemistry of the StAR and P450 scc proteins was performed using antisera prepared against synthetic peptide for both proteins and estradiol-17beta (E2); testosterone (T) and 11-ketotestosterone (11-KT) tissue levels were performed using enzyme immunoassay (EIA). Our data show that EE2 produced time- and concentration-specific effects on the StAR protein, P450 scc, P450 arom isoforms, and IGF-2 gene expressions in salmon gonadal tissues. Cellular expression of the StAR and P450 scc proteins was mainly demonstrated in follicular cells of the oocyte membrane, showing time- and EE2 concentration-dependent differences in staining intensities. Tissue levels of E2, T, and 11-KT in salmon were differentially modulated by EE2 in a time- and concentration-specific manner. Although an apparent negative relationship between E2 and T that reflected aromatization of T to E2 was observed at day 3 postexposure, T and 11-KT showed an apparent concentration-dependent effect after EE2 exposure at day 14. The consistencies between our data at day 14 postexposure suggest that the EE2 modulates steroidogenesis by targeting the initial and rate-limiting step that involves the StAR protein. In general, these findings show that the synthetic pharmaceutical endocrine disruptor and ubiquitous environmental pollutant also produce variations in key gonadal steroidogenic and growth-regulating pathways. These effects and the hormonal imbalance reported in the present study may have potential consequences for the vitellogenic process and overt fecundity in teleosts.

Steroid hormone (estrogens and androgens) synthesis and regulation involve a large number of enzymes and potential biochemical pathways. In the context of these biochemical pathways, it is believed that the true rate-limiting step in acute steroid production is the movement of cholesterol across the mitochondrial membrane by the steroidogenic acute regulatory (StAR) protein and the subsequent conversion to pregnenolone by cytochrome P450-mediated side-chain cleavage (P450scc) enzyme. Oocyte development is a complex process that is triggered by the maturation-promoting factor (MPF) involving cyclin-B as a regulatory factor. In the present study, we evaluated the endocrine effects of 17alpha-methyltestosterone (MT) on steroidogenic pathways of Atlantic cod (Gadus morhua), using an in vitro previtellogenic oocyte culture technique that is based on an agarose floating method. Tissue was cultured in a humidified incubator at 10 degrees C for 1, 5, 10 and 20 days with different concentrations of the synthetic androgen MT (0 (control), 1, 10, 100 and 1000 microM) dissolved in ethanol (0.3%). Gene expressions for StAR, P450scc, aromatase-alpha (P450aromA) and cyclin-B were detected using validated real-time PCR with specific primer pairs. Cellular localization of the StAR protein and P450scc were performed using the immunohistochemical technique with antisera prepared against synthetic peptide for both proteins. Steroid hormones (estradiol-17beta: E2 and testosterone: T) levels were estimated using enzyme immunoassay. Our data showed significant concentration-specific increase (at day 1 and 5) and decrease (at day 10 and 20) of the StAR mRNA expression after exposure to MT. P450scc expression showed a MT concentration-specific decrease during the exposure periods and cyclin-B mRNA expression was decreased in MT concentration-dependent manner at days 10 and 20 (reaching almost total inhibition after exposure to 1000 microM MT). MT exposure produced variable effects on the P450aromA mRNA expression that can be described as concentration-specific increase (day 1) and decrease (days 5 and 10). Cellular localization of the StAR protein and P450scc demonstrated their expression mainly in ovarian follicular cells. MT produced an apparent concentration-and time-dependent increase of E2 and T levels. Thus, the present study reveals some novel effects of pharmaceutical endocrine disruptor on the development of previtellogenic oocytes in cod. The impaired steroidogenesis and hormonal imbalance reported in the present study may have potential consequences for the vitellogenic process and overt fecundity in teleosts.

Multiple biological effects of tributyltin (TBT) on juvenile salmon have been investigated. Fish were exposed for 7 days to waterborne TBT at nominal concentrations of 50 and 250 microg/L dissolved in dimethyl sulfoxide (DMSO). Hepatic samples were analyzed for gene expression patterns in the hormonal and xenobiotic biotransformation pathways using validated real-time PCR method. Immunochemical and several cytochrome P450 (CYP)-mediated enzyme activity (ethoxyresorufin: EROD, benzyloxyresorufin: BROD, methoxyresorufin: MROD and pentoxyresorufin: PROD) assays were analyzed. Our data show that TBT produced concentration-specific decrease of estrogen receptor-alpha (ERalpha), vitellogenin (Vtg), zona radiata protein (Zr-protein) and increase of estrogen receptor-beta (ERbeta) and androgen receptor-beta (ARbeta) in the hormonal pathway. In the xenobiotic biotransformation pathway, TBT produced apparent increase and decrease at respective low and high concentration, on aryl hydrocarbon receptor-alpha (AhRalpha), AhR nuclear translocator (ARNT) and AhR repressor (AhRR) mRNA. The expression of CYP1A1 and GST showed a TBT concentration-dependent decrease. The AhRbeta, CYP3A and uridine diphosphoglucuronosyl transferase (UGT) mRNA expressions were significantly induced after exposure to TBT. Immunochemical analysis of CYP3A and CYP1A1 protein levels confirmed the TBT effects observed at the transcriptional levels. The effect of TBT on the biotransformation enzyme gene expressions partially co-related but did not directly parallel enzyme activity levels for EROD, BROD, MROD and PROD. In general, these findings confirm previous reports on the endocrine effects of TBT, in addition to effects on hepatic CYP1A isoenzyme at the transcriptional level that transcends to protein and enzymatic levels. The induced expression patterns of CYP3A and UGT mRNA after TBT exposure, suggest the involvement of CYP3A and UGT in TBT metabolism in fish. The effect of TBT on CYP3A is proposed to represent another hormonal effect of TBT not previously reported in any fish or lower vertebrate. The proposed androgenic effect is supported by the observation that TBT also induced ARbeta mRNA expression in a concentration-specific manner. To our knowledge, this is the first study that has simultaneously studied multiple responses after exposure to TBT in fish.

The steroidogenic acute regulatory (StAR) protein and cytochrome P450-mediated cholesterol side-chain cleavage (P450scc) have been localized in most steroidogenic organs and are rapidly synthesized in response to acute tropic hormone stimulation. In this study, we present the development of cod previtellogenic oocyte in vitro culture system, histological and molecular methods for evaluating the effects of endocrine disruptors such as nonylphenol (NP) on steroid hormone levels, the StAR protein and P450scc. In addition, expression pattern of cyclin-B was studied, because of cyclin B's role as an indicator of oocyte growth in fish. The in vitro previtellogenic oocyte culture technique was based on an agarose floating method. Tissue was cultured in a humidified incubator at 10 degrees C for 4, 7, 14 and 21 d with different concentrations of nonylphenol (0 (control), 1, 10, 50 and 100 microM) dissolved in ethanol (0.3%). Gene expressions were detected using validated real-time polymerase chain reaction (PCR) with specific primers. Immunohistochemistry of the StAR protein and P450scc were performed using antisera prepared against synthetic peptide for both proteins. Estradiol-17beta (E2) and 11-ketotestosterone (11-KT) tissue levels were estimated using enzyme immunoassay. Our data show that nonylphenol produced a unique and consistent concentration-specific pattern of modulation for the StAR protein, P450scc and cyclin-B gene expression at day 14 after exposure. This pattern is generally described as increasing from 0 (control) to 1 and 10 microM, and decreased at 50 and 100 microM. The observed changes in the StAR protein, P450scc and cyclin-B levels showed a direct relationship with changes in 11-KT levels at day 14 after exposure. Cellular localization of StAR and P450scc were specific to the follicular cells of previtellogenic oocytes, but with no differences in staining intensities. No significant change in oocyte diameter was observed between the exposure groups. Our data reveal some novel aspects of nonylphenol effects on maturation and oocyte growth in teleosts, suggesting impaired steroidogenesis and hormonal imbalance with potential consequences for the vitellogenic process and overt fecundity.

The objectives of this study were to develop and validate biomarker techniques for aquatic environmental monitoring of endocrine disrupting chemicals (EDCs) in Nigeria aquatic ecosystems, using the Eleyele Lake, which is a major source of domestic water supply to Ibadan and its surrounding towns, as a model aquatic environment and Tilapia guineensis, as a model organism. A total of 55 male and 28 female fish were used for this study. No significant difference in condition factor was observed between the sexes. Evaluation of gross gonadal morphology of the sampled fish showed 33% intersex prevalence in the sampled population, of which respective 71 and 29% were males and females, with visible testis and ovary developing alongside phenotypic females and males. Plasma concentrations of luteinizing hormone (LH), follicle-stimulating hormone (FSH), 11-ketotestosterone (11-KT) and 17β-estradiol (E2) were performed, showing that male fish had significantly higher plasma LH and E2 concentrations, compared to females. Vitellogenin (Vtg) and zona radiata proteins (Zrp) mRNA levels were significantly higher in males, compared to female fish. Contaminant analysis revealed that PCB 81, 123, 138 and 196 were the only PCB congeners detected in sediment and fish muscle (PCB153 in sediment), while dieldrin was the only organochlorine compound (OC) detected in Eleyele sediment. These responses were used in a multivariate analysis, showing that two principal components were extracted and accounted for 74% of total variation in the dataset. The principal component analysis (PCA) showed that male fish variables were positively correlated with PCB congeners 18 and 123, while female fish showed positive correlations with congener 81, 138, 189, 196, indicating sex-specific pattern of association between PCBs concentrations and biomarker expression. In addition, strong positive correlation between male fish and LH, E2, FSH and Vtg was observed, while female fish positively correlated with 11-KT and GSI. These relationships suggest feminization and masculinization of male and female fish, respectively.

The effects of hypoxia and perfluorooctane sulfonamide (PFOSA), given singly and also in combination on endocrine, biotransformation, and oxidative stress responses were investigated in primary culture of salmon hepatocytes. Hypoxia was induced chemically using cobalt chloride (CoCl2) or deferroxamine (DFO). Primary culture of salmon hepatocytes were exposed to either CoCl2 (150 μM) or DFO (100 μM), in the presence or absence of PFOSA at 0, 25, and 50 μM for 24 and 48 h. Changes in transcript levels were analyzed by quantitative (real-time) PCR using gene-specific primers. CYP, catalase, GST, and SOD activities were analyzed spectrophotometrically. The hif-1α mRNA was used to validate cellular hypoxic condition, showing significantly induced transcription after 48-h exposure to DFO and CoCl2. Our data show that transcript levels for endocrine (ERα, Vtg, and Zrp), biotransformation (cyp1a, cyp3a, gst, and udpgt), and oxidative stress responses (catalase (cat), glutathione peroxidase (gpx), and glutathione reductase (gr)) were differentially modulated by PFOSA and hypoxia alone, and these effects were dependent on the response parameters and time of exposure. In combined exposure scenarios, the observed effects were apparently hypoxia-dependent. However, the observed effects at transcript levels were not concomitant with those at functional protein levels, further emphasizing the potential differences that may exist between these biological levels. Biplot of principal component analysis (PCA) showed grouping of response variables after 48 h of exposure. The distribution of observations and variables indicate that PFOSA had little effect on most response variables, while clustering show a unique association between a given hypoxia condition (i.e., CoCl2 or DFO) in combination with PFOSA and transcripts, proteins, or enzyme activities. © Springer Verlag for EuCheMS Division of Chemistry and the Environment. The final publication is available at https://link.springer.com/article/10.1007%2Fs11356-014-3847-y. This is the authors' accepted and refereed manuscript to the article.

It is known that estrogen-like environmental pollutants can feminise gonadal differentiation in frogs resulting in female-biased sex-ratios at metamorphosis. The long-term effects on reproductive function in frogs following larval exposure to pollutants are less known. Amphibian test systems which allow life-cycle studies are therefore needed. The aim of the present study was to characterise long-term estrogenic effects on the reproductive system of the emerging model species Xenopus (Silurana) tropicalis following larval exposure to ethynylestradiol (EE(2)). EE(2) is a synthetic estrogen that has been detected in sewage effluents and in surface waters. Newly hatched tadpoles (Niewkoop Faber (NF) stage 48) were exposed to the nominal EE(2) concentrations 0 (control), 1, 10, and 100 nM (with analytical chemistry support) until complete metamorphosis (NF stage 66). Effects on the reproductive organs were determined in juveniles (1 month after metamorphosis) and in 9-month-old frogs. Larval exposure to EE(2) caused female-biased phenotypic sex-ratios in both juvenile and adult frogs, which is in agreement with previous work on other frog species. Nearly all (97%) of the 63 EE(2)-exposed 9-month-old frogs had ovaries. Histological evaluation of the gonads of the 9-month-old frogs showed that they were sexually mature. Among the adult frogs with ovaries there was a dose-dependent increase in the frequency of individuals lacking oviducts. Adult frogs exposed to 100 nM EE(2) that had ovaries but no oviducts had lower levels of estrogen receptor alpha (ERalpha) mRNA in the brain than control animals and those exposed to 100 nM EE(2) that had ovaries as well as oviducts. EE(2) exposure did not cause any significant changes in ERalpha mRNA levels in the ovaries of the adult frogs. The reduced level of ERalpha mRNA in the brain of individuals with ovaries lacking oviducts suggests an organizing effect of EE(2) on the central nervous system. The results show that transient early life-stage exposure to an environmental pollutant can induce effects on the reproductive organs and the central nervous system that persist into adulthood. Overall, our data suggest that X. tropicalis, which has a shorter generation time than the well-established model species Xenopus laevis, is a suitable model organism for research on developmental reproductive toxicity in anuran species.

The effects on thyroid hormone-dependent gene biomarker responses of the persistent organochlorine pesticide metabolite 1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene (DDE) were investigated after exposure of 4-week-old European common frog (Rana temporaria) (stage 36) tadpoles to two (0.001 and 0.01 ppm) DDE concentrations. Total body weight, total length, and tail length and width increased after 3-day exposure to DDE. Expression patterns of genes encoding for growth hormone, thyroid-stimulating hormone (TSHbeta) and thyroid hormone receptor (TRalpha and TRbeta) isoforms were evaluated in the head, body and tail regions using a validated real-time polymerase chain reaction (PCR) method. The mRNA expression of growth hormone in the body, and TSHbeta in the head showed significant DDE concentration-dependent decreases. While DDE caused variable effects on TRalpha mRNA steady-state, the expression of TRbeta was significantly decreased in the tail by DDE in a concentration-specific manner. The effect of DDE exposure on TRbeta mRNA expression showed a negative correlation with tail length and width during the exposure period. The unique pattern of a DDE-induced decrease of tail TRbeta expression probably reflects the significant role of this thyroid hormone receptor isoform in tail re-absorption and overall metamorphosis in anuran species. Therefore, the present study shows that the evaluation of thyroid hormone-dependent genes may represent quantitative biomarkers of acute exposure to organochlorine pesticides in anuran species during critical developmental periods such as metamorphosis. Given the widespread environmental levels of DDT and its metabolites, these pollutants will remain a subject of concern and their effects on anuran species should be studied in more detail.

The effects of environmental contamination on amphibians are of particular concern because there are reports of declining numbers of species and individuals in most parts of the world during the last 50 years. During the last decade there has been increased focus on the role of persistent organic pollutants as retinoid (vitamin A) disrupters, and their effects on development, growth and sexual differentiation. To study the effects of p,p'-DDE, one of the most persistent metabolites of the pesticide DDT, on retinol homeostasis, we subcutaneously exposed adult male European common frogs (Rana temporaria) to different doses of p,p'-DDE (0, 0.01, 0.1, 1 and 10 mg/kg body mass) and studied the effect of a short term exposure (14 days) on hepatic retinoid levels and CYP26 gene and protein expression. Hepatic retinol concentrations, CYP26 gene and protein levels were analysed using HPLC, quantitative RT-PCR and indirect ELISA, respectively. Our results showed a significant p,p'-DDE dose-specific increase in the hepatic retinol concentration. CYP26 gene and protein expression were reduced in an apparent p,p'-DDE dose-specific manner. The results suggest that p,p'-DDE may interfere with the hepatic metabolism of retinol in adult frogs by decreasing CYP26 expression patterns.

The organ-specific gene expression patterns of P450arom isoforms have been studied in European common frog, Rana temporaria after exposure to DDE, using real-time PCR. Four groups of frogs were subcutaneously injected with DDE at 0.01, 0.1, 1 and 10 mg/kg body weight and one group, serving as the control was injected with pure corn oil. Brain, kidney, testis and liver P450aromA and P450aromB gene expressions were evaluated at day 14 after exposure. P450aromB data show that 0.1 and 10 mg DDE/kg doses caused 76% and 63% (testis) and 79% and 80% reductions, respectively, of mRNA levels. Brain P450aromB mRNA decreased 10% and 34%, respectively, after exposure to 0.01 and 0.1 mg DDE/kg. Thereafter, a 185% and 177% induction response of brain P450aromB was observed in the groups treated with 1 and 10 mg DDE/kg, respectively. In the kidney, 0.01, 0.1, 1 and 10 mg DDE/kg induced a 516%, 178%, 466% and 247% induction of P450aronB mRNA, respectively. P450aromA expression was not quantified in any of the organs. The relative abundance of P450aromB gene expression in different organs is in the order: kidney > brain > liver > testis. The present data suggest potential detrimental effect of organochlorine pesticides (OCs) and their metabolites on the European frog steroidogenic pathways. Given the high persistency in the environment and continued use in developing countries coupled with the tendency for global atmospheric transport, OCs and their metabolites such as DDE will remain a focus of concern both for scientific and societal reasons.

Gene expression patterns for key brain steroidogenic (StAR, P450scc, CYP11beta) and xenobiotic- and steroid-metabolizing enzymes (CYP1A1 and CYP3A) have been investigated in waterborne nonylphenol (5, 15, and 50 microg/ L) treated juvenile Atlantic salmon (Salmo salar), in addition to carrier vehicle (ethanol) exposed fish, sampled at different time intervals (0, 3, and 7 days) after exposure. Gene expression patterns were studied using the quantitative polymerase chain reaction (real-time PCR). Treatment of juvenile salmon with nonylphenol caused significant induction of steroidogenic acute regulatory (StAR) protein mRNA at day 7 postexposure in the group receiving 15 microg of nonylphenol/L. P450scc was first induced in the group treated with 5 microg of nonylphenol/L at day 7; thereafter, an apparent nonylphenol-concentration-dependent decrease in P450scc mRNA was observed. CYP11beta mRNA was significantly induced at day 3 after exposure to 5 betag of nonylphenol/L; thereafter, CYP11beta mRNA levels were inhibited below control levels in the 15 and 50 microg of nonylphenol/L groups at day 3. At day 7, significant induction of CYP11beta mRNA was observed only in the group exposed to 15 microg of nonylphenol/L. For CYP1A1 mRNA, apparent nonylphenol-concentration-dependent decreases were observed at day 7 postexposure. CYP3A mRNA was significantly induced by all nonylphenol exposure concentrations at day 7. When exposed groups were compared, CYP3A transcript was significantly induced between 5 and 15 microg of nonylphenol/ L, and decreased between 15 and 50 microg of nonylphenol/ L. The ethanol control showed a significant reduction of CYP3A mRNA at day 3 postexposure. The present study has demonstrated variations in three key steroidogenic proteins and xenobiotic- and steroid-metabolizing CYP isoenzyme gene transcripts in the brain of nonylphenol-exposed juvenile salmon. Therefore, the present study represents a novel aspect of neuroendocrine effects of nonylphenol in fish not previously demonstrated and should be studied in more detail.

Induction of blood plasma vitellogenin (Vtg) and zona radiata proteins (Zr-proteins) in male and juvenile of oviparous vertebrates was proposed and shown to be sensitive biomarkers for exposure to estrogen mimic. The time- and dose-dependent expression of Vtg and Zr-proteins in nonylphenol (NP) exposed juvenile Atlantic salmon (Salmo salar) is reported. Fish were exposed continuously to waterborne nonylphenol at 5, 15 and 50 μg/L. Blood and surface mucus samples were collected after 3 and 7 days post-exposure. Nonylphenol-induced plasma and surface mucus levels of Vtg and Zr-protein were analysed using immunochemical methods (Western blot and enzyme-linked immunosorbent assay; ELISA). Both Vtg and Zr-protein levels in plasma and surface mucus showed similar and parallel nonylphenol-induced expression patterns after waterborne nonylphenol exposure and in a concentration- and time-dependent manner. Zr-proteins were significantly induced at the lowest concentration of nonylphenol after 3 and 7 days of exposure both in plasma and in surface mucus. We conclude that the detection of Vtg and Zr-proteins directly in the surface mucus of fish, and the correlation of these values with plasma protein biomarker values in xenoestrogen-treated fish represents a sensitive non-invasive system for the detection of these known endocrine disruptor biomarkers. The demonstration of detectable Vtg and Zr-protein levels from surface mucus is a potential biomarker for estrogenic compounds, and their presence should be considered as an improvement in the methods for detection of endocrine disrupting chemicals (EDCs) and related pollutants in the environment.

The short-term effects of the commercial PBDE flame retardant mixtures Penta-BDE and cta-BDE on the expression of cytochrome P450 1A (CYP1A), vitellogenin (Vtg) and zona radiata proteins (Zrp) were investigated in juvenile salmon (Salmo salar). For this purpose, groups of fish were dosed twice (oral intake at days I and 4) with 10 and 50 mg/kg body weight of both commercial mixtures. The fishes were sacrificed at day 7 (n = 5 for each group) and 14 (n = 6 for each group), and blood, liver, fillet, and brain were collected. Blanks and positive controls were also part of the experiment. The expressions of Vtg, Zrp, and CYPIA were measured with several techniques (EROD, ELISA, Western, Northern and Slot Blot). The values in the groups of fish treated with Penta-BDE or Octa-BDE did not significantly differ from the reference group for any of the parameters tested. In contrast, the positive control groups treated with estradiol-17beta for Vtg and Zrp expression, and beta-naphthoflavone for CYP1A expression did show a significant response, indicating the potential sensitivity of the fishes for the parameters measured. Since the results of the chemical analyses showed concentrations of a number of PBDE congeners in liver, fillet, and brain that were about three orders of magnitude above those of fish from the North Sea, it is concluded that the short-term toxicity of both commercial PBDE mixtures for these endpoints was low.

The relative importance of environmental hypoxia due to global climate change on organismal ability to adapt to chemical insult and/or mechanisms of these responses is not well understood. Therefore, we have studied the effects of combined exposure to perfluorooctane sulfonamide (PFOSA) and chemically induced hypoxia on membrane lipid profile and homeostasis. Primary salmon hepatocytes were exposed to PFOSA at 0, 25 and 50 mM singly or in combination with either cobalt chloride (CoCl2: 0 and 150 mM) or deferroxamine (DFO: 0 and 100 mM) for 24 and 48 h. CoCl2 and DFO were used to induce cellular hypoxia because these two chemicals have been commonly used in animal experiments for this purpose and have been shown to increase hypoxia-inducible factor 1-alpha (HIF-1a) and vascular endothelial growth factor (VEGF) levels. Fatty acid (FA) profiles were determined by GC-MS, while gene expression patterns were determined by quantitative PCR. Hypoxic condition was confirmed with time-related increases of HIF-1a mRNA levels in CoCl2 and DFO exposed cells. In general, significant alterations of genes involved in lipid homeostasis were predominantly observed after 48 h exposure. Gene expression analysis showed that biological responses related to peroxisome proliferation (peroxisome proliferatoractivated receptors (PPARs) and acyl coenzyme A (ACOX)) and FA desaturation (D5- and D6-desaturases: FAD5 and FAD6, respectively) and elongation (FAE) were elevated slightly by single exposure (i.e. either PFOSA, CoCl2 or DFO exposure alone), and these responses were potentiated in combined exposure conditions. Principal component analysis (PCA) showed a clustering of peroxisome proliferation responses at transcript levels and FA desaturation against membrane FAs levels whose changes were explained by PFOSA and chemically induced hypoxia exposures. Overall, our data show that most of the observed responses were stronger in combined stressor exposure conditions, compared to individual stressor exposure. In general, our data show that hypoxia may, singly or in combination with PFOSA produce deleterious health, physiological and developmental consequences through the alteration of membrane lipid profile in organisms. © 2014 Olufsen et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Thyroid hormones (triiodothyronine, T3; and thyroxine, T4) play significant roles in development, metamorphosis, metabolism, homeostasis, cellular proliferation, and differentiation, for which the effects are mediated through thyroid hormone receptors (TRα and TRβ). Similarly, the insulin-like growth factor (IGF) is involved in growth and development through regulation of somatic growth. This study was designed to examine the effects of the dioxin-like 3,3',4,4'-tetrachlorobiphenyl (PCB-77) on responses related to growth and thyroid hormone system in eyed eggs and yolk-sac larvae of Atlantic salmon. Salmon eggs were continuously exposed to two waterborne concentrations of PCB-77 (1 or 10 ng/L) over a period of 50 d covering hatching and through yolk-sac absorption stages. Sampling was performed regularly throughout the exposure period and at different time intervals. Gene expression patterns were performed on whole-body homogenate at age 500, 548, 632, 674, and 716 dd (dd: day degrees) using quantitative polymerase chain reaction (PCR). Total T3 (TT3) and total T4 (TT4) were measured using radioimmunoassay (RIA). Data showed that 10 ng PCB-77 increased dioiodinase 2 (Dio2) at 500 dd and both PCB-77 concentrations decreased dio2 expression at 548 dd. PCB-77 elevated cellular TT3 at 500 dd and was lowered at 548 dd only at 10 ng. Otherwise, time-related reduction was not affected by PCB-77 exposure as observed for the rest of the exposure period. For TT4, 1 ng PCB-77 produced a rise at 500 dd, and an apparent concentration decrease at 548 dd, before a total inhibition at 632 dd. The IGF-1 and IGF-1R were variably affected by PCB-77. For IGF-2, PCB-77 produced a concentration-dependent increase at 548 dd, and thereafter an elevation (1 ng) and fall (10 ng) at 632 dd. TRβ mRNA demonstrated PCB-77 related increases during the exposure period, and this effect returned to control levels at 716 dd. For TRα, a rise was noted only after exposure to 10 ng PCB-77 at 500 dd. Overall, the present study demonstrates some possible growth and developmental consequences following exposure to PCB-77 during early life stages of Atlantic salmon.

In the aquatic environments, the predicted changes in water temperature, pO2 and pCO2 could result in hypercapnic and hypoxic conditions for aquatic animals. These conditions are thought to affect several basic cellular and physiological mechanisms. Yet, possible adverse effects of elevated CO2 (hypercapnia) on teleost fish, as well as combined effects with emerging and legacy environmental contaminants are poorly investigated. In this study, juvenile Atlantic cod (Gadus morhua) were divided into groups and exposed to three different water bath PFOS exposure regimes (0 (control), 100 and 200 μg L−1) for 5 days at 1 h/day, followed by three different CO2-levels (normocapnia, moderate (0.3%) and high (0.9%)). The moderate CO2 level is the predicted near future (within year 2300) level, while 0.9% represent severe hypercapnia. Tissue samples were collected at 3, 6 and 9 days after initiated CO2 exposure. Effects on the endocrine and biotransformation systems were examined by analyzing levels of sex steroid hormones (E2, T, 11-KT) and transcript expression of estrogen responsive genes (ERα, Vtg-α, Vtg-β, ZP2 and ZP3). In addition, transcripts for genes encoding xenobiotic metabolizing enzymes (cyp1a and cyp3a) and hypoxia-inducible factor (HIF-1α) were analyzed. Hypercapnia alone produced increased levels of sex steroid hormones (E2, T, 11-KT) with concomitant mRNA level increase of estrogen responsive genes, while PFOS produced weak and time-dependent effects on E2-inducible gene transcription. Combined PFOS and hypercapnia exposure produced increased effects on sex steroid levels as compared to hypercapnia alone, with transcript expression patterns that are indicative of time-dependent interactive effects. Exposure to hypercapnia singly or in combination with PFOS produced modulations of the biotransformation and hypoxic responses that were apparently concentration- and time-dependent. Loading plots of principal component analysis (PCA) produced a significant grouping of individual scores according to the exposure scenarios at day 6 and 9. Overall, the PCA analysis produced a unique clustering of variables that signifies a positive correlation between exposure to high PFOS concentration and mRNA expression of E2 responsive genes. Notably, this pattern was not evident for individuals exposed to PFOS concentrations in combination with elevated CO2 scenarios. To our knowledge, the present study is the first of its kind, to evaluate such effects using combined exposure to a perfluoroalkyl sulfonate and elevated levels of CO2 saturation, representative of future oceanic climate change, in any fish species or lower vertebrate. © 2014. This is the authors’ accepted and refereed manuscript to the article. This manuscript version is made available under the CC-BY-NC-ND 4.0 license http://creativecommons.org/licenses/by-nc-nd/4.0/

Zonagenesis (zona radiata protein synthesis) and vitellogenesis (yolk protein synthesis) are two reproductive responses that are integral aspects of fish oogenesis. This study examines the responses of eggshell zona radiata proteins (Zrp) and vitellogenin (Vtg) to five environmental pollutants; 4-nonylphenol (NP) and o,p'-DDT [both at 25 mg/kg], lindane (gamma-HCH) [10 mg/kg], a technical PCB mixture (Aroclor 1254; A1254) and bisphenol A (BPA) [both at 5 mg/kg] in juvenile Atlantic salmon (Salmo salar). Fish were given intraperitoneal injections of o,p'-DDT, gamma-HCH, A1254 or BPA; singly, in combination with NP, and as a cocktail of all five chemicals, and later compared to NP-treated and untreated fish. In a separate experiment, fish were exposed to BPA in a dose-response manner (1, 5, 25 and 125 mg/kg fish). Based on previous studies, blood and liver samples were collected 2 weeks after injection. Zrp and Vtg levels were analyzed in plasma using immunoblotting and enzyme-linked immunosorbent assay. Liver cytochrome P4501A was analyzed by 7-ethoxyresorufin O-deethylase (EROD) activity. NP caused pronounced elevations in plasma Zrp and Vtg levels. In comparison, when NP was given in combination with gamma-HCH, Vtg levels was significantly reduced, compared to NP treatment alone. Using o,p'-DDT, A1254 and BPA, significant elevations of plasma Zrp and Vtg were seen when chemicals were given in combination with NP, but not when administered by themselves. An apparent dose-response induction of Vtg and Zrp levels were observed in BPA treated juvenile salmon. In immunoblots, one component of molecular weight approximating the Zrp-beta was detected when either o,p'-DDT, gamma-HCH, A1254 or BPA were given singly. A1254 significantly induced hepatic EROD activity when administered alone. However, when given as a mixture with all the other xenobiotics, reduction of EROD activity was observed. The data suggest a pattern of xenobiotics action which may complicate assessment of their reproductive effects. Zrp (the beta monomer) was more responsive to the xenoestrogens than Vtg, and provides a sensitive means of detecting exposure to environmental estrogens.

The in vivo estrogenic potency of zearalenone (ZEA), a mycotoxin produced by different strains of Fusarium fungi, and its metabolites (alpha- and beta-zearalenol), have been studied in fish. Estrogenicity was evaluated using an in vitro competitive receptor binding assay and in vivo induction of vitellogenesis and zonagenesis, two estrogen receptor (ER)-mediated responses that are integral aspects of fish oogenesis. The ER binding affinities of alpha-zearalenol and ZEA in rainbow trout (Oncorhynchus mykiss) were approximately 1/150 and 1/300 to that of estradiol, respectively. Juvenile salmon (Salmo salar) were exposed to a single intraperitoneal injection of ZEA, alpha-zearalenol and beta-zearalenol (each at 1 and 10 mg/kg) and compared to fish injected with estradiol-17 beta (E2; 5 mg/kg) and controls. Using indirect enzyme-linked immunosorbent assay (ELISA) with homologous antibodies, a dose-dependent induction of vitellogenin (Vtg) and eggshell zona radiata proteins (Zr-proteins) were observed 7 days after exposure to ZEA and alpha-zearalenol. beta-Zearalenol did not elevate plasma Vtg levels, but a non-significant elevation of plasma Zr-proteins levels was observed at the highest dose (10 mg/kg). Generally, alpha-zearalenol and ZEA possess estrogenic potencies that are approximately 50% compared to that of E2, and their order of estrogenic potency (in both in vitro receptor competitive binding and in vivo induction of Vtg and Zr-proteins levels) is: alpha-zearalenol > ZEA > beta-zearalenol. Our results show that blood plasma analysis of Vtg and Zr-proteins levels provides a suitable in vivo fish model for assessing the estrogenic potencies of ZEA and its metabolites.

A liver complementary DNA expression library from Atlantic salmon (Salmo salar) pretreated with estradiol-17beta (E2) was constructed and screened with antibodies raised against salmon eggshell (zona radiata) proteins. Two clones, SalZr2_19 and SalZr2_23 were sequenced and shown to encode proteins of approximately 50 kDa. SalZr2_23 contains 12 octamer sequence lpqr/kpa/vq repeats also found in SalZr2_19, but only twice. Alignment reveals that the two salmon sequences are similar to piscine zona radiata proteins and mammalian zona pellucida proteins. Several transcripts ranging from 2.3 to 12 kb appeared in liver extracts of E2-treated fish. Juvenile fish treated with E2 or 4-nonylphenol showed strong induction of zona radiata protein. The use of egg envelope transcriptional and translational induction in male or juvenile fish as a biological marker of environmental estrogens is discussed.

Juvenile rainbow trout were given intraperitoneal (IP) injections of (i) octylphenol (OP) and butylbenzylphthalate (BBP) or (ii) octylphenol and estradiol (E). Both pairs of compounds were administered singly and in combination, at two different dose levels. Modulation of the hepatic estradiol receptor (ER) binding capacity and induction of hepatic zona radiata proteins (Zrp) were utilised as indicators of estrogenic effect. In treatment (i) OP caused a significant (approx 2-fold) upregulation of the ER and a significant (approx 3-5 fold) increase in Zrp at both doses compared to control, vehicle-injected fish. In contrast, no effect on the ER was observed in BBP-treated fish, but plasma Zrp levels were significantly reduced. No synergistic effect was observed on ER modulation or Zrp induction in fish receiving OP and BBP in combination. In treatment (ii) both OP and E caused a significant (approx 2-fold) upregulation of the ER and a significant (approx 6-7 fold) increase in plasma Zrp at both dose levels compared to controls. The effect of OP and E in combination, on both ER modulation and induction of Zrp, was additive and not synergistic.

Induction of vitellogenin (Vtg) in males and juveniles of oviparous vertebrates has been used as a biomarker for xenoestrogens. Recently, we have demonstrated that synthesis of eggshell zona radiata proteins (Zrp) or zonagenesis is an integral aspect of fish oogenesis (Oppen-Berntsen et al. (1994) Journal of Experimental Zoology 268, 59–70), and that Zrp synthesis is a sensitive biomarker for nonylphenol (Arukwe et al. (1997) Environmental Health Perspective 105, 418–422). This study compares the responses of Vtg and Zrp in plasma of juvenile Atlantic salmon (Salmo salar) treated with 4-nonylphenol (NP) and o,p′-DDT (both at 25 mg kg−1, singly and in combination). Validated ELISA and immunoblot analysis show that Vtg and Zrp respond significantly stronger to NP treatment alone and in combination with o,p′-DDT compared to control and o,p′-DDT treatment alone. However, a slight reduction in NP-induced Zrp levels was indicated when NP was injected in combination with o,p′-DDT.

Hepatic microsomal biotransformation reactions with xenobiotic and steroid substrates have been investigated in 4-nonylphenol (NP: 1, 5, 25, and 125 mg/kg body weight)-treated juvenile Atlantic salmon (Salmo salar), in addition to control and estradiol-17β (5 mg/kg, positive control)-treated fish. Treatment of juvenile salmon with NP caused an initial increase and an apparent dose-dependent decrease in progesterone 6β-, 16α, and 17α-hydroxylase activities in liver microsomes. 7-Ethoxyresorufin O-deethylase and UDP-glucuronosyltransferase activities were also reduced. Plasma levels of estradiol-17β (E 2 ) were lowered 24-43% as a result of NP treatment. Immunochemical analysis of CYPIA, CYP2K-like, and CYP3A-like proteins showed 18%, 47%, and 30% reductions in enzyme-linked immunosorbent assay absorbance levels, respectively, in the groups treated with 125 mg NP/kg fish. The group treated with E 2 also showed similar reductions. In summary, the present study has demonstrated variations in steroid hydroxylases, cytochrome P450 isozymes, and conjugating enzyme levels in NP-treated juvenile salmon. These results represent a novel aspect of NP effects not previously demonstrated with an environmental estrogen in any fish species or lower vertebrate.

Sexual differentiation of the hepatic cytochrome P450 system was characterized in 2-year-old farmed turbot (Scophthalmus maximus L.) during their first spawning period (January–September). The fish were kept in tanks supplied with continuously flowing seawater (34.5%, ppt) at a constant temperature of 16°C and natural photoperiod (60°N). Sampling of liver samples (n = 4–6) was performed once every month for 9 months. Pronounced sex differences were recorded in the activities of 7-ethoxyresorufin O-deethylase (EROD), 7-ethoxycoumarin O-deethylase (ECOD), and NADPH-cytochrome P450 reductase during spawning (May–July). EROD activity in female fish decreased gradually towards the onset of ovulation in May–July to rise again in the postspawning period. The decrease correlated with increasing gonadosomatic index and estradiol17β (E2) levels in plasma. Immunochemical detection of CYP1A (58 kDa), CYP2K-like (47 and 52 kDa), and CYP3A-like (58 and 60 kDa) proteins in Western blotting, and ELISA showed higher protein levels in male compared to female fish from April/May-June, and significant differences were observed in June (CYP2K-like also in April and May). Analysis of monthly variations within sexes during the reproductive cycle shows significant monthly changes in all parameters in both female and male fish. Both CYP2K- and CYP3A-like protein levels were significantly elevated in male fish during spawning in June. To study the induction response during spawning, β-naphthoflavone (BNF) 75 mg/kg body weight) was administered intraperitoneally to both sexes in June. BNF caused a significant increase in EROD and ECOD activities and CYP1A protein levels but had no effect on NADPH-cytochrome P450 reductase activity or CYP2K-like/CYP3A-like proteins. This study documents, for the first time in any fish species or lower vertebrate, the sexual differentiation in the liver of three different CYP subfamilies during sexual maturation and spawning. J. Exp. Zool. 277:313–325, 1997. © 1997 Wiley-Liss, Inc.

Induction of vitellogenin (Vtg) in oviparous vertebrates has been used as a biomarker of response for environmental oestrogens. This study reports the cellular localization of oestrogen- and xenoestrogen-induced Vtg synthesis in the liver of juvenile Atlantic salmon (Salmo salar). Paraffin-embedded liver sections were incubated with homologous monoclonal antibody against Atlantic salmon Vtg. Following intraperitoneal (ip) exposure of fish to estradiol-17beta E2; 5 mg kg-1 or 4-nonylphenol NP; 125 mg kg-1, Vtg induction was primarily demonstrated immunohistochemically in the cytoplasm of hepatocytes, endothelial cells and within hepatic sinusoids. Vtg staining of hepatocytes was not evenly distributed, as there was a high degree of polarization toward the sinusoid. The intensity of positive Vtg staining was stronger in the liver sections of E2-treated fish, compared with NP-treated fish. Hepatocytes of E2-, NP- and vehicle (control)-treated fish showed normal cellular structures, thus showing no evidence of histopathological changes. In parallel, indirect enzyme-linked immunosorbent assay (ELISA) and immunoblot analysis of plasma Vtg levels show significant induction of Vtg in E2- and NP-treated fish, as compared with untreated (control) fish. The present study demonstrates the applicability of immunohistochemistry in studies of cellular structures, processes and responses of fish exposure to oestrogen and oestrogen-mimicking compounds.

In the present preliminary study, we used a gas chromatography–mass spectroscopy (GC-MS) method to identify and quantify steroid hormones, their precursors and metabolites in whole body of red palm weevil (RPW) R. ferrugineus adults. We identified a total of seven steroids by single ion monitoring mode (SIM) analysis and compared them to the National Institute of Standards and Technology (NIST) library. The steroids include: dehydroepiandrosterone (DHEA), estrone, estradiol-17β, testosterone, progesterone, cortisol and cholesterol, whereas pregnenolone, pregnan-20-one-17-hydroxy and corticosterone were not detected. This study shows that some invertebrate groups seem to use partially or totally comparable hormones to vertebrates and produce vertebrate-type steroids with functional roles. However, some steroids of the endocrine system of invertebrates are still lacking or yet to be identified in most phyla.

The present study has investigated the effects of domperidone (DOM), a dopamine 2 receptor antagonist, on plasma steroid hormone levels and reproductive performances of a female tropical marine finfish, Waigieu seaperch (Psammoperca waigiensis), with potential for cultivation in Vietnam. We showed that oral treatment of DOM during early stage of the reproductive cycle had no significant effects on the maturation and reproductive performances of the female fish, while plasma steroid hormone (testosterone (T), 11-ketotestosterone (11-KT), 17β-estradiol (E2) and progesterone (P)) levels were modulated based on month, DOM dose and the individual hormones measured. Overall, these findings suggest that DOM may not be needed for the induction of maturation and spawning of this species under aquaculture conditions. The data in the present study are significant in highlighting practical efforts for reducing drug use, production costs and for a sustainable aquaculture in a developing country such as Vietnam.

We have studied the effects of dioxin-like 3,3′,4,4′-tetrachlorobiphenyl (PCB-77) on developmental effects related to angiogenesis and osteogenesis during early life-stages of salmon. Larvae were kept at 6 °C and continuously exposed to waterborne PCB-77 (1 or 10 ng/L) initiated at the egg stage or 416-day degrees (dd) and throughout yolk-sac stage (716 dd) and for a total duration of 50 days (or 300 dd). Gene transcription analysis was performed on whole larvae total RNA at 548, 632, 674 and 716 dd using real-time PCR. Bone morphogenetic protein (bmp2 and bmp4), transforming growth factor β (TGF-β), estrogen receptors (ERα and ERβ), runx2, sox9 and collagen type 2 alpha 1 (col2a1) and vascular endothelial growth factor (VEGF) genes were studied. Effect on VEGF gene transcription was related to observation of heart rate, arrhythmia and anemia, demonstrating effects on vascular system development. Alizarine-red staining and quantification of ossified bone structures showed that PCB-77 produced concentration-dependent increases in the rate of osteogenic tissue formation. PCB-77 produced increases in col2a1 and runx2 transcription with subsequent induction of chondrogenesis and osteogenesis, respectively. The transcription of TGF-β gene was associated with ERβ transcription. Transcripts of AhR gene battery were differentially modulated by PCB-77 and these effects were dependent on concentration and larval age. Evidence of vascular system disruption by PCB-77 was observed as cardiac edema, anemia and arrhythmia in exposed individuals and as decreased level of VEGF gene transcription at early age. In general, our data indicate that PCB-77 produced developmental effects related to angiogenesis and osteogenic differentiation and disruption of vascular system development.

In this study, we have investigated the effect of perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA) on endocrine signalling, growth and development in Atlantic salmon ( Salmo salar ) embryos and larvae. Expression of genes related to the hypothalamic–pituitary–thyroid (HPT) axis, growth-hormone/insulin-like growth factor (GH/IGF) axis and the steroid hormone axis were used as indicators of endocrine disruption. We also studied bone development in larvae, both by observing skeletal structure formation and by investigating expression of genes involved in ossification process. Atlantic salmon embryos, kept in plastic tanks at 5–7 °C, were exposed to 100 μg/L PFOA or PFOS from egg stage for a period of 52 days, followed by one-week recovery period. Sampling was performed at day 21, 35, 49 and 56 representing age 549, 597, 679 and 721 dd (dd or day degrees = number of days × temperature in degree Celsius: °C). Note that day 56 or 721 dd is the end of the 1-week recovery period. Larvae were divided into designated head and body regions for the purpose of gene expression analysis, except for genes that regulate ossification that were analyzed in whole larvae. Expression of thyroid receptor α and β (TRα and TRβ), thyroid-stimulating hormone β (TSHβ), T 4 outer-ring deiodinase (T 4 ORD), growth hormone (GH), insulin-like growth factor-I and II (IGF-I and II), insulin-like growth factor I receptor (IGF-IR), and estrogen receptor α and β (ERα and ERβ) were investigated using quantitative PCR. Both PFOS and PFOA exposure produced non-significant alterations in larvae weight (except after the recovery period when a decrease was observed), while larvae length was unaffected. PFOS and PFOA exposure produced body- and head region-specific alterations in expression of all the investigated gene transcripts. Expression of IGF-I and IGF-IR paralleled that of GH, indicating that perturbation of GH expression is a possible end point for disruption of the GH–IGF axis. We did not observe developmental changes related to angiogenesis, ossification and chondrogenesis after exposure to PFOS and PFOA. Transcriptional abnormalities may serve as indicators of chronic exposure, although the concrete mechanisms causing the observed effects remain ambiguous. The implications of these findings for the complete lifecycle, including other developmental and/or reproductive damage, are areas of future study.

In the present study, groups of juvenile Atlantic salmon (Salmo salar) were fed gelatine capsules containing fish-food spiked with PFOA or PFOS (0.2 mg kg(-1) fish) and solvent (methanol). The capsules were given at days 0, 3 and 6. Blood, liver and whole kidney samples were collected prior to exposure (no solvent control), and at days 2, 5, 8 and 14 after exposure (Note: that day 14 after exposure is equal to 7d recovery period). We report on the differences in the tissue bioaccumulation patterns of PFOS and PFOA, in addition to tissue and compound differences in modulation pattern of biotransformation enzyme genes. We observed that the level of PFOS and PFOA increased in the blood, liver and kidney during the exposure period. Different PFOS and PFOA bioaccumulation patterns were observed in the kidney and liver during exposure- and after the recovery periods. Particularly, after the recovery period, PFOA levels in the kidney and liver tissues were almost at the control level. On the contrary, PFOS maintained an increase with tissue-specific differences, showing a higher bioaccumulation potential (also in the blood), compared with PFOA. While PFOS and PFOA produced an apparent time-dependent increase in kidney CYP3A, CYP1A1 and GST expression, similar effects were only temporary in the liver, significantly increasing at sampling day 2. PFOA and PFOS exposure resulted in significant decreases in plasma estrone, testosterone and cortisol levels at sampling day 2, and their effects differed with 17α-methyltestostrerone showing significant decrease by PFOA (also for cholesterol) and increase by PFOS. PFOA significantly increased estrone and testosterone, and no effects were observed for cortisol, 17α-methyltestosterone and cholesterol at sampling day 5. Overall, the changes in plasma steroid hormone levels parallel changes in CYP3A mRNA levels. Given that there are no known studies that have demonstrated such tissue differences in bioaccumulation patterns with associated differences in toxicological responses in any fish species or lower vertebrate, the present findings provide some potential insights and basis for a better understanding of the possible mechanisms of PFCs toxicity that need to be studied in more detail.

We investigated variables related to thyroid, vitamin A and calcitriol homeostasis, immune function and tumour development in ringed seals (Phoca hispida) from the polluted Baltic Sea and a less polluted reference location at Svalbard, Norway. We also examined the relationships between the biological variables and the concentrations of persistent organic pollutants (POPs) and their hydroxylated (OH) metabolites. Our data show higher plasma concentrations of free triiodothyronine (T3), and ratios of free and total T3 in Baltic seals as compared to Svalbard seals. Baltic seals had also higher hepatic mRNA expressions of deiodinase-I, thyroid hormone receptor beta, retinoic acid receptor alpha, growth hormone receptor and interleukin-1beta compared to Svalbard seals. Levels of plasma retinol were lower in the Baltic seals as compared to Svalbard seals. No geographical difference was observed for other thyroid hormone levels and hepatic retinoid levels. Ratios of free and total T3 were positively correlated to OH-POPs in plasma. The results of the present study suggest that endocrine homeostasis may be affected by contaminant and metabolite exposure in the Baltic ringed seals with respect to circulating hormones and retinol and hepatic mRNA expressions. In addition, OH-POPs may putatively produce the disruption of thyroid hormone transport in plasma.