R848 Is Involved in the Antibacterial Immune Response of Golden Pompano (Trachinotus ovatus) Through TLR7/8-MyD88-NF-κB-Signaling Pathway.

R848 is an imidazoquinoline compound that is a specific activator of toll-like receptor (TLR) 7/8 and is often used in immunological research in mammals and teleosts. However, the immune responses initiated by R848 through the TLR7/8 pathway in response to bacterial infection remain largely unexplored in teleosts. In the current study, we investigated the antibacterial response and the participating signaling pathway initiated by R848 in golden pompano (Trachinotus ovatus). We found that R848 could stimulate the proliferation of head kidney lymphocytes (HKLs) in a dose-dependent manner, enhance the survival rate of HKLs, and inhibit the replication of bacteria in vivo. However, these effects induced by R848 were significantly reduced when chloroquine (CQ) was used to blocked endosomal acidification. Additionally, an in vivo study showed that R848 strengthened the antibacterial immunity of fish through a TLR7/8 and Myd88-dependent signaling pathway. A cellular experiment showed that Pepinh-MYD (a Myd88 inhibitor) significantly reduced the R848-mediated proliferation and survival of HKLs. Luciferase activity analysis showed that R848 enhanced the nuclear factor kappa B (NF-κB) activity, whereas this activity was reduced when CQ and Pepinh-MYD were present. Additionally, when an NF-κB inhibitor was present, the R848-mediated pro-proliferative and pro-survival effects on HKLs were significantly diminished. An in vivo study showed that knockdown of TLR7, TLR8, and Myd88 expression in golden pompano via siRNA following injection of R848 resulted in increased bacterial dissemination and colonization in fish tissues compared to that of fish injection of R848 alone, suggesting that R848-induced antibacterial immunity was significantly reduced. In conclusion, these results indicate that R848 plays an essential role in the antibacterial immunity of golden pompano via the TLR7/8-Myd88-NF-κB- signaling pathway.

Estrogen-responsive gene networks in the teleost liver: What are the key molecular indicators?

An overarching goal of environmental genomics is to leverage sensitive suites of markers that are robust and reliable to assess biological responses in a range of species inhabiting variable environments. The objective of this study was to identify core groups of transcripts and molecular signaling pathways that respond to 17alpha-ethylinestadiol (EE2), a ubiquitous estrogenic contaminant, using transcriptome datasets generated from six independent laboratories. We sought to determine which biomarkers and gene networks were those most robust and reliably detected in multiple laboratories. Six laboratories conducted microarray analysis in pieces of the same liver from male fathead minnows exposed to ∼15ng/L EE2 for 96h. There were common transcriptional networks identified in every dataset. These included down-regulation of gene networks associated with blood clotting, complement activation, triglyceride storage, and xenobiotic metabolism. Noteworthy was that more than ∼85% of the gene networks were suppressed by EE2. Leveraging both these data and those mined from the Comparative Toxicogenomics Database (CTD), we narrowed in on an EE2-responsive transcriptional network. All transcripts in this network responded ∼±5-fold or more to EE2, increasing reliability of detection. This network included estrogen receptor alpha, transferrin, myeloid cell leukemia 1, insulin like growth factor 1, insulin like growth factor binding protein 2, and methionine adenosyltransferase 2A. This estrogen-responsive interactome has the advantage over single markers (e.g. vitellogenin) in that these entities are directly connected to each other based upon evidence of expression regulation and protein binding. Thus, it represents an interacting functional suite of estrogenic markers. Vitellogenin, the gold standard for estrogenic exposures, can show high individual variability in its response to estrogens, and the use of a multi-gene approach for estrogenic chemicals is expected to improve sensitivity. In our case, the coefficient of variation was significantly lowered by the gene network (∼67%) compared to Vtg alone, supporting the use of this transcriptional network as a sensitive alternative for detecting estrogenic effluents and chemicals. We propose that screening chemicals for estrogenicity using interacting genes within a defined expression network will improve sensitivity, accuracy, and reduce the number of animals required for endocrine disruption assessments.

Cimetidine disrupts the renewal of testicular cells and the steroidogenesis in a hermaphrodite fish.

The importance of histamine in the physiology of the testis in mammals and reptiles has been recently shown. Histamine receptors (Hrs) are well conserved in fish and are functional in several fish species. We report here for the first time that histamine and the mRNA of Hrh1, Hrh2 and Hrh3 are all present in the gonad of the hermaphrodite teleost fish gilthead seabream. Moreover, cimetidine, which acts in vitro as an agonist of Hrh1 and Hrh2 on this species, was intraperitoneally injected in one and two years old gilthead seabream males. After three and five days of cimetidine injection, we found that this compound differently modified the gonadal hrs transcript levels and affects the testicular cell renewal and the gene expression of steroidogenesis-related molecules as well as the serum steroid levels. Our data point to cimetidine as a reproductive disruptor and elucidate a role for histamine in the gonad of this hermaphrodite fish species through Hr signalling.

Functional characterization of rainbow trout (Oncorhynchus mykiss) Abcg2a (Bcrp) transporter.

ABCG2 (BCRP - breast cancer resistance protein) belongs to the ATP-binding cassette (ABC) superfamily. It plays an important role in the disposition and elimination of xeno- and endobiotics and/or their metabolites in mammals. Likewise, the protective role of ABC transporters, including Abcg2, has been reported for aquatic organisms. In our previous study we have cloned the full gene sequence of rainbow trout (Oncorhynchus mykiss) Abcg2a and showed its high expression in liver and primary hepatocytes. Based on those insights, the main goal of this study was to perform a detailed functional characterization of trout Abcg2a using insect ovary cells (Spodoptera frugiperda, Sf9) as a heterologous expression system. Membrane vesicles preparations from Sf9 cells were used for the ATPase assay determinations and basic biochemical properties of fish Abcg2a versus human ABCG2 have been compared. A series of 39 physiologically and/or environmentally relevant substances was then tested on interaction with trout Abcg2a and human ABCG2. Correlation analysis reveals highly similar pattern of activation and inhibition. Significant activation of trout Abcg2a ATPase was observed for prazosin, doxorubicine, sildenafil, furosemid, propranolol, fenofibrate and pheophorbide. Pesticides showed either a weak activation (malathione) or strong (endosulfan) to weak (chlorpyrifos, fenoxycarb, DDE) inhibition of trout Abcg2a ATPase while the highest activation was obtained for benzo(a)pyrene, curcumine and testosterone. In conclusion, data from this study offer the first characterization of fish Abcg2a, reveal potent interactors among physiologically or environmentally relevant substances and point to similarities regarding strengths and interactor preferences between human ABCG2 and fish Abcg2a.

Acute ammonia toxicity in crucian carp Carassius auratus and effects of taurine on hyperammonemia.

The four experimental groups were carried out to test the response of crucian carp Carassius auratus to ammonia toxicity and taurine: group 1 was injected with NaCl, group 2 was injected with ammonium acetate, group 3 was injected with ammonium acetate and taurine, and group 4 was injected with taurine. Fish in group 2 had the highest ammonia and glutamine contents, and the lowest glutamate content in liver and brain. Serum superoxide dismutase (SOD), glutathione (GSH) activities, red cell count (RBC), white cell count (WBC), lysozyme (LYZ) activity, complement C3 content of fish in group 2 reflected the lowest, but malondialdehyde content was the highest. Importantly, serum SOD and GSH activites, RBC, WBC, and LYZ activity, C3, C4 and total immunoglobulin contents of fish in group 3 were significantly higher than those of fish in group 2. This study indicates that ammonia exerts its toxic effects by interfering with amino acid transport, inducing ROS generation, leading to malondialdehyde accumulation and immunosuppression of crucian carp. The exogenous taurine could mitigate the adverse effect of high ammonia level on fish physiological disorder.

In vitro nuclear receptor activity and in vivo gene expression analysis in Murray-Darling rainbowfish (Melanotaenia fluviatilis) after short-term exposure to fluoxetine.

Fluoxetine (FLX) is one of numerous pharmaceuticals found in treated municipal wastewater discharged to the environment. In the present study, we investigated the effects of short-term (96h) waterborne FLX exposure (1µg/L or 100µg/L) on the expression of selected genes in brain, liver, and gonads of female Murray-Darling rainbowfish (Melanotaenia fluviatilis), a small-bodied teleost of ecotoxicological relevance in the Australasia region. Plasma 17ß-estradiol (E2) levels were also determined. In the brain, no significant changes in mRNA levels were observed for the selected genes. In ovaries, 100µg/L FLX caused a 10-fold downregulation of aromatase A (cyp19a1a) mRNA and a 4-fold upregulation of estrogen receptor α (esr1) mRNA levels. In liver, mRNA levels for vitellogenin A (vtga) and choriogenin L (chgl) were downregulated by 50-fold and 18-fold compared with controls, respectively, in response to 100µg/L FLX. Concentrations of E2 in plasma were significantly lower than controls in response to 100µg/L FLX. This could be attributable to a decrease in estrogen biosynthesis as a result of the observed downregulation of cyp19a1a mRNA. To establish whether the observed changes in gene expression could be explained by the modulation of selected nuclear receptors by FLX, we employed panel of reporter gene assays in agonistic and antagonistic modes. Apart from minor activation of ERα after exposure to high concentrations (5µM), FLX did not activate or inhibit the nuclear receptors tested. Further study is required to determine whether the observed downregulation of ovarian aromatase expression and liver estrogen-regulated genes also occurs at environmentally relevant FLX concentrations over longer exposure periods.

ABC transporters and xenobiotic defense systems in early life stages of rainbow trout (Oncorhynchus mykiss).

Embryos of oviparous fish, in contrast to (ovo) viviparous species, develop in the aquatic environment, and therefore need solute transport systems at their body surfaces for maintaining internal homeostasis and defending against potentially harmful substances. We hypothesized that solute transporters undergo changes in tissue distribution from the embryo to the larval stage. We therefore studied the mRNA profiles of eight ABC transporters (abcb1a, abcb1b, abcc1, abcc2, abcc3, abcc4, abcc5, abcg2) and three solute carriers (oatp1d, putative oatp2 putative, mate1) in different body regions (head, yolk sac epithelium, abdominal viscera, skin/muscles) of developing rainbow trout. Additionally, we investigated mRNA levels of phase I (cyp1a, cyp3a) and phase II (gstp, putative ugt1, putative ugt2) biotransformation enzymes. The study covered the developmental period from the eleuthero-embryo stage to the first-feeding larval stage (1-20days post-hatch, dph). At 1dph, transcripts of abcc2, abcc4, abcg2, cyp3a, gstp, putative mate1, and putative oatp2 occurred primarily in the yolk sac epithelium, whereas at later stages expression of these genes was predominantly observed in the abdominal viscera. The functional activity of ABC transporters in fish early life stages was assessed by rhodamine B accumulation assays. Finally, we investigated the potential impact of xenobiotics (clotrimazole, clofibric acid) on the ABC and biotransformation systems of trout early life stages. While clofibric acid had no effect, clotrimazole lead to an increased rhodamine B accumulation. The results provide evidence that the transition from the eleuthero-embryo to the larval stage is accompanied by a major alteration in tissue expression of ABC transporters.

Developmental toxicity of endocrine disruptors in early life stages of zebrafish, a genetic and embryogenesis study.

Endocrine disrupting compounds (EDCs) are capable of interfering with the endocrine system and are increasingly widespread in the aquatic environments. In the present study, zebrafish (Danio rerio) embryos and larvae were used to assess how EDCs may interfere with embryogenesis. Therefore, zebrafish embryos were exposed to 17α-ethinylestradiol (EE2: 0.4, 2, 4 and 20 ng/L), genistein (Gen: 2, 20, 200 and 2000 ng/L) and fadrozole (Fad: 2, 10, 50 and 250 µg/L), between 2 and 144 h post-fertilization (hpf). Somite development, heartbeat, malformations, mortality and hatching rates were evaluated. In parallel, the expression patterns of hormone receptors (esr1, esr2a, esr2b and ar) and apoptotic pathways related genes (p53 and c-jun) were determined using quantitative real-time PCR. Results showed that EE2, Gen and Fad caused a higher mortality and also malformations in larvae compared with control. A significant toxic effect was observed in the heartbeat rate, at 144 hpf, in larvae exposed to EE2 and Fad. QPCR revealed alterations in the expression levels of all the evaluated genes, at different time points. esr1 and c-jun genes were upregulated by EE2 and Gen exposure while the expression of esr2a, esr2b and ar genes was downregulated. Fad exposure decreased esr1, p53 and c-jun expression levels. This study shows a toxic effect of EE2, Gen and Fad to vertebrate embryogenesis and a relation between hormones action and apoptosis pathways.

Control of transcriptional repression of the vitellogenin receptor gene in largemouth bass (Micropterus salmoides) by select estrogen receptors isotypes.

The vitellogenin receptor (Vtgr) plays an important role in fish reproduction. This receptor functions to incorporate vitellogenin (Vtg), a macromolecule synthesized and released from the liver in the bloodstream, into oocytes where it is processed into yolk. Although studies have focused on the functional role of Vtgr in fish, the mechanistic control of this gene is still unexplored. Here we report the identification and analysis of the first piscine 5' regulatory region of the vtgr gene which was cloned from largemouth bass (Micropterus salmoides). Using this putative promoter sequence, we investigated a role for hormones, including insulin and 17ß-estradiol (E2), in transcriptional regulation through cell-based reporter assays. No effect of insulin was observed, however, E2 was able to repress transcriptional activity of the vtgr promoter through select estrogen receptor subtypes, Esr1 and Esr2a but not Esr2b. Electrophoretic mobility shift assay demonstrated that Esr1 likely interacts with the vtgr promoter region through half ERE and/or SP1 sites, in part. Finally we also show that ethinylestradiol (EE2), but not bisphenol-A (BPA), represses promoter activity similarly to E2. These results reveal for the first time that the Esr1 isoform may play an inhibitory role in the expression of LMB vtgr mRNA under the influence of E2, and potent estrogens such as EE2. In addition, this new evidence suggests that vtgr may be a target of select endocrine disrupting compounds through environmental exposures.

Melatonin effects on Fundulus heteroclitus reproduction.

This study aimed to investigate the effects of two different doses (100nM (M1) and 1µM (M2)) of exogenous melatonin on the reproductive capacity of Fundulus heteroclitus. Eight days of melatonin exposure significantly increased the fecundity and embryo survival of F. heteroclitus only in the M2 group compared with the control; the hatching rate was unaffected. Moreover, increases in the local expression of the melatonin receptor (mtnr) gene during follicle maturation were found; however, there were no differences between the experimental groups. Furthermore, in vitro melatonin-treated follicles showed a significantly higher germinal vesicle break down percentage compared with the control, while SDS-PAGE showed no difference in the electrophoretic pattern of the major yolk proteins. Nevertheless, densitometry revealed a greater intensity of the 118-, 95- and 40-kDa components in groups treated with melatonin. Finally, Fourier transform infrared microspectroscopy was applied to classify the different stages of oocyte development (Stages I-II, III and IV) on the basis of their macromolecular composition. The effects induced by melatonin on oogenesis were investigated by comparing vibrational spectra of females exposed to melatonin with those of controls. Changes to the Amide I band, corresponding to an increase in ß-structure, were found in oocytes of females exposed to the highest melatonin dose. These results highlight the positive role of melatonin, which is able to enhance the reproductive capacity of F. heteroclitus. Further studies are in progress to better explain the molecular mechanisms by which melatonin treatment affects reproduction in this marine species.

Effects of sexual steroids on the expression of foxl2 in Gobiocypris rarus.

Gobiocypris rarus is an emerging fish model for aquatic toxicology in China as it is sensitive to environmental hormone disruptors. Exogenous sex steroids can affect sex differentiation and the expression of sex-related genes. Foxl2, a member of forkhead-box transcription factor family, is the key gene for ovary development and its mutation causes the blepharophimosis ptosis epicanthus inversus syndrome in human. We find that two foxl2 genes exist in fish genome, one is foxl2, and the other is foxl2b. Here, we reported the isolation and expression of foxl2 in G. rarus. G. rarus foxl2 cDNA is 1700bp in length with a 921bp of open reading frame encoding 306 amino acids containing the typical FH-domain. Semi-quantitative RT-PCR revealed its predominant expression in the eye, brain, gill and gonads. Moreover, the expression level in the ovary was significantly higher than that in the testis. Quantitative RT-PCR showed that foxl2 was up regulated after treatment with estradiol and was down regulated with 2-methyl-testosterone. These results suggested that Foxl2 plays an important role in female development of G. rarus, foxl2 mRNA expression is regulated by downstream sex hormones, and foxl2 can be used as a molecular indicator monitoring the environmental endocrine disruptors.

Hepatic in vitro toxicity assessment of PBDE congeners BDE47, BDE153 and BDE154 in Atlantic salmon (Salmo salar L.).

The brominated flame retardant congeners BDE47, BDE153 and BDE154 are among the congeners accumulating to the highest degree in fish. In order to gain knowledge about the toxicological effects of PBDEs in fish, microarray-based transcriptomic and 2D-DIGE/MALDI-TOF/TOF proteomic approaches were used to screen for effects in primary Atlantic salmon hepatocytes exposed to these congeners alone or in combination (PBDE-MIX). A small set of stress related transcripts and proteins were differentially expressed in the PBDE exposed hepatocytes. The PBDE-MIX, and BDE153 to a lesser degree, seems to have induced metabolic disturbances by affecting several pathways related to glucose homeostasis. Further, effects on cell cycle control and proliferation signal pathways in PBDE-MIX-exposed hepatocytes clearly suggest that the PBDE exposure affected cell proliferation processes. CYP1A was 7.41- and 7.37-fold up-regulated in hepatocytes exposed to BDE47 and PBDE-MIX, respectively, and was the only biotransformation pathway affected by the PBDE exposure. The factorial design and PLS regression analyses of the effect of the PBDE-MIX indicated that BDE47 contributed the most to the observed CYP1A response, suggesting that this congener should be incorporated in the toxic equivalent (TEQ) concept in future risk assessment of dioxin-like chemicals. Additionally, a significant up-regulation of the ER-responsive genes VTG and ZP3 was observed in cells exposed to BDE47 and PBDE-MIX. Further analyses suggested that BDE47 and BDE154 have an estrogenic effect in male fish. The data also suggested an antagonistic interaction between BDE153 and BDE154. In conclusion, this study shows that PBDEs can affect several biological systems in Atlantic salmon cells, and demonstrates the need for more studies on the simultaneous exposure to chemical mixtures to identify combined effects of chemicals.

Brain proteome alterations of Atlantic cod (Gadus morhua) exposed to PCB 153.

Polychlorinated biphenyls (PCBs) are still widespread environmental pollutants that bioaccumulate and biomagnify in the aquatic food chains despite the ban on their production. They constitute a class of 209 possible congeners with different chlorination pattern of the biphenyl ring structure resulting in many different toxicities and mechanisms of toxicity. The neurotoxicity of PCBs is relatively poorly understood, and biomarkers for their neurotoxic effects are lacking. We have carried out a proteomic analysis of brain tissue from Atlantic cod (Gadus morhua) exposed to 2,2',4,4',5,5'-hexachlorobiphenyl (PCB 153, ortho-substituted and non-coplanar), a previously demonstrated neurotoxic congener and the most prevalent congener in biological samples. The fish received 0, 0.5, 2 and 8 mg/kg PCB 153 by intraperitoneal injection, half of the dose on the first day and the second half after one week, and were exposed for two weeks in total. Using a 2-DE approach we found 56 protein spots to be 20% or more (≤ 0.8-fold or ≥ 1.2-fold) significantly different between at least one of the three PCB 153-exposed groups and the control group, and 27 of these were identified by MALDI-TOF MS and MS/MS. Approximately 80% of the differentially regulated proteins may be associated with a non stressor-specific response and/or have previously been classified as notoriously differentially regulated in 2-DE/MS based proteomics studies, such as alterations/responses in energy metabolism, cytoskeleton, protein synthesis, protein degradation (ubiquitin-proteasome system), cellular growth, cycle and death (14-3-3 protein), and (surprisingly) axon guidance (dihydropyrimidinase-like 2 (=collapsin response mediator protein 2, CRMP-2)). The six remaining affected proteins include the strongest up-regulated protein, pyridoxal kinase (essential for synthesis of neurotransmitters such as dopamine, serotonin and GABA), nicotinamide phosphoribosyl-transferase (involved in protection against axonal degeneration) and protein phosphatase 1 (controls brain recovery by synaptic plasticity). The last three of these six proteins (deltex, Rab14 and sorting nexin 6) may preliminarily identify involvement of the Notch signaling pathway and endosomal function in PCB 153-induced neurotoxicity. Our findings constitute novel clues for further research on PCB 153 mode of action in brain, and a proper selection of proteins may, following validation, be applicable in a panel of biomarkers for aquatic environmental monitoring.

Differential effects of 17ß-estradiol and 11-ketotestosterone on the endocrine stress response in zebrafish (Danio rerio).

Sexually dimorphic stress responses are present in species across all vertebrate taxa and it has been suggested that these effects are mediated by circulating sex steroids. While a few species of fish have been identified as having a sexually dimorphic stress response, there is conflicting evidence as to the effects of sex steroids on the stress axis. In this study, we tested whether zebrafish exhibit a sexually dimorphic cortisol stress response and whether 17ß-estradiol (E2) or 11-ketotestosterone (11KT) modulate the activity of the hypothalamic-pituitary-interrenal (HPI) axis. To accomplish this, we quantified the whole body cortisol response to a physical stressor, cortisol release in vitro, and the expression of key HPI axis regulating genes of control and E2- or 11KT-exposed zebrafish. Under control conditions no dimorphisms in the HPI axis were apparent at rest or in response to a standardized stressor. In contrast, E2-exposure blunted the cortisol response of male fish in vivo and in vitro and as well as corticotropin-releasing factor (crf) expression in the pre-optic area (POA) of the brain. While the expression of some interrenal genes was suppressed by E2-exposure, these changes occurred in both male and female zebrafish. 11KT-exposure increased whole-body cortisol of males at rest and vortex-exposed females, but had no impact on the rate of cortisol synthesis in vitro or on POA crf expression. Therefore, while we found no evidence that zebrafish exhibit a sexually dimorphic cortisol stress response, both E2 and 11KT can modulate the activity of the HPI axis in this species and do so via different mechanisms.

Disrupting actions of bisphenol A and malachite green on growth hormone receptor gene expression and signal transduction in seabream.

Environmental estrogen could mimic natural estrogens thereby disrupting the endocrine systems of human and animals. The actions of such endocrine disruptors have been studied mainly on reproduction and development. However, estrogen could also affect the somatotropic axis via multiple targets such as growth hormone (GH). In the present study, two endocrine disruptors were chosen to investigate their effects on the expression level and signal transduction of growth hormone receptor (GHR) in fish. Using real-time PCR, it was found that exposure to both the estrogenic (bisphenol A) and anti-estrogenic (malachite green) compounds could attenuate the expression levels of GHR1 and GHR2 in black seabream (Acanthopagrus schlegeli) hepatocytes. The expression level of IGF-I, the downstream effector of GHR activation in the liver, was decreased by bisphenol A but not by malachite green. Luciferase reporter assay of the beta-casein promoter was used to monitor GHR signaling in transfected cells. In the fish liver cell line Hepa-T1, both GHR1 and GHR2 signaling were attenuated by bisphenol A and malachite green. This attenuation could only occur in the presence of estrogen receptor, indicating that these agents probably produce their actions via the estrogen receptor. Results of the present study demonstrated that estrogenic or anti-estrogenic compounds could down-regulate the somatotropic axis in fish by affecting both the gene expression and signaling of GHR. In view of the increasing prevalence of these compounds in the environment, the impact on fish growth and development both in the wild and in aquaculture would be considerable.

In vivo measurement of protein functional changes.

Conformational changes in proteins are fundamental to all biological functions. In protein science, the concept of protein flexibility is widely used to describe protein dynamics and thermodynamic properties that control protein conformational changes. In this study, we show that urea, which has strong sedative potency, can be administered to fish at high concentrations, and that protein functional changes related to anesthesia induction can be measured in vivo. Ctenopharyngodon idellus (the grass carp) has two different types of N-methyl d-aspartate (NMDA) receptors, urea-insensitive and urea-sensitive, which are responsible for the heat endurance of fish. The urea-sensitive NMDA receptor showed high protein flexibility, the gamma aminobutyric acid (GABA) receptor showed less flexibility, and the protein that is responsible for ethanol anesthesia showed the lowest flexibility. The results suggest that an increase in protein flexibility underlies the fundamental biophysical mechanisms of volatile general anesthetics.

Halquinol modulated growth, physiology, and protein profile and halquinol residue withdrawal study in the Indian major carp Catla catla (Hamilton).

The aim of the present study was to evaluate the effect of halquinol, an antimicrobial used as a growth promotor in poultry, on the fresh water fish Catla catla in terms of growth promotion, protein profile, and physiology as the rate of oxygen consumption. A synergic increment in the free amino acid level and total protein concentration suggested enhanced anabolic metabolism resulting in weight gain. When compared with an untreated control group, fishes treated with 0.1% halquinol (T1) showed a higher weight gain than those treated with 0.2% halquinol (T2). Variations in the rate of oxygen consumption among the three groups (control, T1, T2) expressed the physiological response of the animals toward the chemical along the time factor. After 7 days of treatment, the absence of halquinol revealed by post-withdrawal residual HPLC studies suggests its biosafety.

Negative roles of salt in gelation properties of fish protein isolate.

Salt effect on gelling properties of fish protein isolate (FPI) prepared by acid- and alkali-aided extraction was investigated. Acid- or alkali-extracted FPI formed significantly better gel texture with 0% NaCl than with 3% NaCl. Texture properties of acid- or alkali-extracted FPI decreased as NaCl content increased, especially at 2% to 3% salt. Contrarily, salt significantly promoted texture qualities of conventional surimi gels. The effect was highlighted when they were subjected to low temperature setting. The myofibrillar proteins in FPI were not solubilized when NaCl was added, perhaps due to protein aggregation caused by acid or alkali extraction. FPI solubility, however, was not closely related to their texture properties. Cold setting did not promote texture properties of FPI gels as much as conventional surimi gels. Acid-extracted gels gave the best color properties.

Effects of 17alpha-ethynylestradiol on EROD activity, spiggin and vitellogenin in three-spined stickleback (Gasterosteus aculeatus).

The three-spined stickleback (Gasterosteus aculeatus) has quantifiable biomarkers of exposure to estrogens (vitellogenin), androgens (spiggin) and aryl hydrocarbon receptor (AhR) agonists (EROD activity) and is therefore a promising test species for biomonitoring of reprotoxic chemicals in aquatic environments. In this study we evaluated the effects of 17alpha-ethynylestradiol (EE(2)) on EROD activity, induction of vitellogenin and spiggin, hepatosomatic index (HSI), ovarian somatic index (OSI) and nephrosomatic index (NSI). Adult male and female three-spined sticklebacks were exposed to concentrations of 0-170 ng EE(2)/l (measured concentrations) in a flow-through system for 21 days. Exposure to 170 ng EE(2)/l resulted in a significant 8- and 9-fold induction of gill EROD activity in males and females, respectively. In livers, EROD activity expressed in relation to microsomal protein content was suppressed due to a significant increase in microsomal protein content. Hepatic EROD activity per se expressed as picomol/min was not affected by exposure to EE(2). The lowest observed effect concentration for induction of vitellogenin in males was 53.7 ng EE(2)/l. In females, vitellogenin levels were significantly higher in those exposed to 170 ng EE(2)/l compared to controls. Spiggin production was significantly inhibited and NSI lower in males exposed to 170 ng EE(2)/l. In both females and males LSI was significantly higher in fish exposed to 170 ng EE(2)/l than in controls. In females exposed to 170 ng EE(2)/l, OSI was significantly lower and NSI higher than controls. The observed results from this study show that a synthetic estrogen can affect the well-known biomarker of exposure for dioxin-like compounds, EROD activity, and further that this response can differ between tissues. These findings are important for interpretation of biomonitoring data.