Toxicity screening of produced water extracts in a zebrafish embryo assay.

Produced water is the largest effluent discharge from oil and gas/condensate production facilities in the North Sea. There is concern that contaminants originating from the reservoir and chemicals used in the production process may affect marine organisms. Developmental toxicity of extractable organic compounds in produced water effluents from oil and gas/condensate production platforms in the Norwegian sector of the North Sea was assessed in a temporal and spatial manner using zebrafish (Danio rerio) embryos. Large-scale solid-phase extraction (SPE) and on-column fractionation of water-soluble fraction (WSF) and an oil/particulate fraction was used in a rapid screening bioassay for embryotoxicity. Exposure to produced water extracts increased rate of mortality and reduced pigmentation and heart rate, as well as delaying time to hatch. The oil/particulate fraction was 10-fold less toxic than WSF, indicating that toxicity was predominantly produced by moderately polar and bioavailable compounds. Large spatial and temporal variation in produced water toxicity was observed, displaying considerable variability in the reservoir, oil well, and effluent composition over time. The noted toxicity did not correlate well with either reported produced water composition or parameters such as total hydrocarbons, thus challenging chemical measurements as a reliable source of information for predicting complex effects. Although embryotoxicity was observed following exposure to the extracts, dilution and transformation of produced water in the recipient are expected to rapidly reduce the concentrations of compounds in the effluents to levels below the thresholds of observed effects.

Endocrine modulation in Atlantic cod (Gadus morhua L.) exposed to alkylphenols, polyaromatic hydrocarbons, produced water, and dispersed oil.

Effluent from oil production activities contains chemicals that are suspected of inducing endocrine disruption in fish. In this study, Atlantic cod (Gadus morhua L.) were exposed to mixtures of low- and medium-molecular-weight alkylphenols (AP) (methyl- to heptylphenol), polycyclic aromatic hydrocarbons (PAH), diluted produced water, and dispersed oil for 15 d in a flow-through exposure system. Condition index (CI), hepatosomatic index (HSI), gonadosomatic index (GSI), concentration of the estrogenic biomarker vitellogenin (Vtg), and modulation of the total sex steroid-binding capacity in plasma were determined to assess whether these mixtures were capable of interfering with endocrine-regulated physiological processes in Atlantic cod. No marked differences in plasma Vtg levels were found between control and exposed groups of either males or females, possibly due to high intergroup variances and low sample numbers. An apparent numerical increase in the number of male and female fish with high plasma Vtg levels was, however, observed in some exposure groups compared to control. This purported weak estrogenic effect was several orders of magnitude lower than that observed for potent estrogens and suggested that the levels of estrogen receptor (ER) agonists were low. Exposure of female fish to a mixture of dispersed oil and a mixture of AP, PAH, and dispersed oil led to upregulation of the plasma total sex steroid-binding capacity, indicating interference with the normal blood steroid transport. No significant effects were seen for CI, HSI, and GSI, suggesting that the endocrine-disrupting potential was not sufficient to elicit effects on general physiological conditions and gonad development during this short exposure period.

Why is the multiple stressor concept of relevance to radioecology?

Purpose: A number of nuclear and radiological sources have contributed or are still contributing to the release of a series of stressors such as radionuclides in combination with trace metals and even organic chemicals. To assess the impact of mixed contamination, a limited number of stressors are usually evaluated one by one. We have therefore evaluated the benefit of using the multiple stressor concept focusing on key topics within radioecology such as the source term and deposition, ecosystem interactions and exposure, biological uptake and effects including adverse outcome pathways (AOPs), as a basis for assessing impact and risk. Materials and methods: We have extracted information from highly relevant scientific articles (e.g. Web of Science) describing multiple stressor exposure experiments where at least one stressor is ionizing radioactivity such as low dose gamma radiation or radionuclides (e.g. fission or activation products; uranium and daughter nuclides). In addition, experiences obtained during recent years at the author´s laboratories are included in the discussions, especially when it comes to speciation issues, combined effects, risk estimates, and AOPs. Results: The multiple stressor concept is not only related to the total concentration of a series of radionuclides and other stressors released from one source or from different sources, but also related to the presence of different physico-chemical forms of individual radionuclides. Due to interactions, the ecosystem transfer to biota is dynamic, and the application of toxicokinetics and toxicodynamics considerations seems most useful. Although there is a limited number of articles dealing with multiple stressors in which ionizing radiation is included as one of the stressors, deviation from concentration (dose) additive responses should probably be expected, especially under field conditions where additional abiotic as well as biotic interactions take place Conclusions: Following severe nuclear events, releases can be described as a massive multiple stressor exposure containing radionuclides combined with other stressors. Thus, advanced characterization technologies are needed for parameterization of the mixed stressor source term as input to ecosystem transport, dose, and impact models. To reduce uncertainties in assessments of these complex exposures, advanced technologies and computational efforts to link internal distributions to responses are also needed to understand how multiple stressor exposure could affect toxicokinetics and toxicodynamics, and why combined exposures could depart from additivity along the source-adverse outcome continuum.

The partial pressure of oxygen affects biomarkers of oxidative stress in cultured rainbow trout (Oncorhynchus mykiss) hepatocytes.

Oxidative stress, the imbalance between production of reactive oxygen species and the cellular detoxification of these reactive compounds, is believed to be involved in the pathology of various diseases. Several biomarkers for oxidative stress have been proposed to serve as tools in toxicological and ecotoxicological research. Not only may exposure to various pro-oxidants create conditions of cellular oxidative stress, but hyperoxic conditions may also increase the production of reactive oxygen species. The objective of the current study was to determine the extent to which differences in oxygen partial pressure would affect biomarkers of oxidative stress in a primary culture of hepatocytes from rainbow trout (Oncorhynchus mykiss). Membrane integrity, metabolic activity, levels of total and oxidized glutathione (tGSH/GSSG) was determined, as well as mRNA expression levels of the antioxidant enzymes superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), glutathione reductase (GSSG-R), gamma-glutamyl-cystein synthetase (GCS) and thioredoxin (TRX). The results show that different biomarkers of oxidative stress are affected when the cell culture is exposed to atmospheric oxygen, and that changes such as increased GSSG content and induction of GSSG-R and GSH-Px can be reduced by culturing the cells under lower oxygen tension. Oxygen tension may thus influence results of in vitro based cell research and is particularly important when assessing parameters in the antioxidant defence system. Further research is needed to establish the magnitude of this effect in different cellular systems.

Uptake rates of alkylphenols, PAHs and carbazoles in semipermeable membrane devices (SPMDs) and polar organic chemical integrative samplers (POCIS).

Passive sampling devices provide a useful contribution to the monitoring of contaminants in the aquatic environment. However, calibration data needed for the calculation of water concentrations from sampler accumulations are restricted to a limited number of compound classes. Thus uptake of a range of alkylated phenols (AP), polycyclic aromatic hydrocarbons (PAH) and carbazoles was determined for semipermeable membrane devices (SPMDs) and polar organic chemical integrative samplers (POCIS) using a flow through exposure system. Sampling rates ranged from 0.02 to 0.26 l d(-1) for POCIS and 0.02 to 13.83 l d(-1) for SPMDs. Observed SPMD uptake was also compared to that predicted by an empirical model including the use of performance reference compounds (PRCs). Predicted sampling rates did not differ by more than a factor of 1.3 from experimental values for PAH, providing further evidence that the PRC approach can be successfully used to determine in situ sampling rates for these compounds. Experimental sampling rates for AP in SPMDs were, however, much lower than predicted. This discrepancy was too large to be explained by small uncertainties in the calibration system or in the calculations. Based on these data we conclude that while hydrophobic AP are accumulated by SPMDs their partitioning cannot be predicted from their logK(ow) using current methods. Due to this lower than expected uptake, sampling rates were only higher in SPMDs than POCIS in the range of logK(ow)>5.0. Simultaneous deployment of both sampler types allows the study of compounds with a broad range of physicochemical properties.

Estrogenicity of alkylphenols and alkylated non-phenolics in a rainbow trout (Oncorhynchus mykiss) primary hepatocyte culture.

Alkylphenols act as estrogen mimics by binding to and transactivating estrogen receptors (ERs) in fish. In the present study, activation of ER-mediated production of the estrogenic biomarker vitellogenin (vtg) in a primary culture of rainbow trout (Oncorhynchus mykiss) hepatocytes was used to construct a structure-activity relationship for this ubiquitous group of aquatic pollutants. The role of alkyl chain length and branching, substituent position, number of alkylated groups, and the requirement of a phenolic ring structure was assessed. The results showed that most alkylphenols were estrogenic, although with 3-300 thousand times lower affinity than the endogenous estrogen 17beta-estradiol. Mono-substituted tertiary alkylphenols with moderate (C4-C5) and long alkyl chain length (C8-C9) in the para position exhibited the highest estrogenic potency. Substitution with multiple alkyl groups, presence of substituents in the ortho- and meta-position and lack of a hydroxyl group on the benzene ring reduced the estrogenic activity, although several estrogenic alkylated non-phenolics were identified. Co-exposures with the natural estrogen 17beta-estradiol led to identification of additional estrogenic compounds as well as some anti-estrogens. A combination of low affinity for the ER and cytotoxicity was identified as factors rendering some of the alkylphenols non-estrogenic in the bioassay when tested alone.

Toxicogenomic responses in rainbow trout (Oncorhynchus mykiss) hepatocytes exposed to model chemicals and a synthetic mixture.

As more salmon gene expression data has become available, the cDNA microarray platform has emerged as an appealing alternative in ecotoxicological screening of single chemicals and environmental samples relevant to the aquatic environment. This study was performed to validate biomarker gene responses of in vitro cultured rainbow trout (Oncorhynchus mykiss) hepatocytes exposed to model chemicals, and to investigate effects of mixture toxicity in a synthetic mixture. Chemicals used for 24h single chemical- and mixture exposures were 10 nM 17alpha-ethinylestradiol (EE2), 0.75 nM 2,3,7,8-tetrachloro-di-benzodioxin (TCDD), 100 microM paraquat (PQ) and 0.75 microM 4-nitroquinoline-1-oxide (NQO). RNA was isolated from exposed cells, DNAse treated and quality controlled before cDNA synthesis, fluorescent labelling and hybridisation to a 16k salmonid microarray. The salmonid 16k cDNA array identified differential gene expression predictive of exposure, which could be verified by quantitative real time PCR. More precisely, the responses of biomarker genes such as cytochrome p4501A and UDP-glucuronosyl transferase to TCDD exposure, glutathione reductase and gammaglutamyl cysteine synthetase to paraquat exposure, as well as vitellogenin and vitelline envelope protein to EE2 exposure validated the use of microarray applied to RNA extracted from in vitro exposed hepatocytes. The mutagenic compound NQO did not result in any change in gene expression. Results from exposure to a synthetic mixture of the same four chemicals, using identical concentrations as for single chemical exposures, revealed combined effects that were not predicted by results for individual chemicals alone. In general, the response of exposure to this mixture led to an average loss of approximately 60% of the transcriptomic signature found for single chemical exposure. The present findings show that microarray analyses may contribute to our mechanistic understanding of single contaminant mode of action as well as mixture effects, but that its use in screening of complex environmental samples will need to be further evaluated.

Interaction of estrogen mimics, singly and in combination, with plasma sex steroid-binding proteins in rainbow trout (Oncorhynchus mykiss).

The plasma sex steroid-binding protein (SBP) is believed to be involved in regulating circulating endogenous sex steroids as well as cellular signal transduction to nuclear steroid receptors in sex steroid-sensitive tissues. In this study, a variety of estrogen mimics (EMs), which may contribute to the endocrine disrupting effects observed in fish, were tested for the ability to interact with the rainbow trout plasma sex steroid-binding protein (rtSBP) either singly or in binary combinations. The EMs ethynylestradiol, diethylstilbestrol, 4-hydroxytamoxifen, genistein, zearalenone, 4-t-octylphenol, bisphenol A and o,p'-DDT were all able to displace 1,2,4,6,7-[3H]estradiol from the sex steroid-binding site at the rtSBP (K(d)=2.1+/-0.5 nM, B(max)=2963+/-303 fmol estradiol/mg protein) in a dose-dependent and competitive manner. The plastizicer n-butyl benzyl phthalate only displayed weak binding affinity for the rtSBP, whereas the pesticide dieldrin was not able to compete for the high affinity estradiol-binding sites in plasma. None of the compounds tested was able to clearly promote the binding of the others when given in combination, indicating that synergy did not occur at the ligand-SBP binding level. The rtSBP binding affinity for EMs ranged over several orders of magnitude, but were consistently lower than those for the endogenous sex steroids (about 10(2)-10(6) less potent). The results suggest that the presence of rtSBP may potentially modulate the bioavailability of EMs to estrogen receptors relative to each other and to the endogenous sex steroids themselves. Since the binding of the endogenous ligands is reversible, SBP-bound estrogens (or androgens) potentially may also become displaced by potent EMs.

Acetylcholine esterase inhibitors in effluents from oil production platforms in the North Sea.

Inhibition of acetylcholine esterase (AChE) activity is a biomarker for the exposure to neurotoxic compounds such as organophosphates and is intimately associated with the toxicity of several pesticides. In the present study, the AChE inhibiting potential of organic extracts of production water (produced water) from oil and gas production platforms in the Norwegian sector of the North Sea was determined in an in vitro bioassay based on commercially available purified AChE from the electric organ of Electrophorus electricus (L.). The results from the studies show that produced water contains a combination of AChE inhibiting compounds and compounds stimulating AChE enzymatic activity. The AChE inhibition was predominantly caused by unidentified aromatic compounds in the oil/particulate fraction of produced water, whereas polar compounds in both the water soluble and oil/particulate fraction of produced water caused an apparent stimulation of AChE activity. Substrate saturation studies with fixed concentrations of produced water extracts confirmed that the inhibition occurred in a non-destructive and competitive manner. The concentrations of AChE inhibitors (7.9-453 ng paraoxon-equivalents L⁻¹, 2.2-178 µg dichlorvos-equivalents L⁻¹) were in many cases found to be several orders of magnitude higher than background levels. The findings demonstrate that produced water contains potentially neurotoxic compounds and suggest that further laboratory studies with fish or field studies in the vicinity of oil production facilities are highly warranted.

Acute and sub-lethal effects in juvenile Atlantic salmon exposed to low µg/L concentrations of Ag nanoparticles.

Silver nanoparticles (Ag-NP) are components in numerous commercial products and are discharged into the environment in quantities that are largely unknown. In the present study, juvenile Atlantic salmon were exposed to 1, 20, and 100 µg/L (48 h, static renewal) of a commercially available Ag-NP colloidal suspension in natural (soft) lake water. A solution of AgNO(3) containing 20 µg/L Ag(I) ions was also included to discriminate the effect of NPs from that of ionic silver. Furthermore, the commercial Ag-NP suspension was compared to an in-house synthesised colloidal NP suspension prepared from AgNO(3) and NaBH(4) in citrate buffer. The size distribution of Ag in all exposure solutions was characterised by 0.22 µm filtration and 10 kDa hollow fibre cross-flow ultrafiltration in combination with ICP-MS. All exposures were characterised by a relatively high proportion of Ag-NP in the colloidal size fraction 3-220 nm. For assessment of biological effects, acute toxicity, gill histopathology, blood plasma parameters (Na, Cl, glucose, haemoglobin), and gene expression of a selection of gill biomarkers were measured. Results showed that the gills accumulated Ag in all exposure groups apart from the fish exposed to 1 µg/L Ag-NP. Accumulated Ag caused concentration-dependent response increases in general stress markers such as plasma glucose and gill gene expression of heat shock protein 70. Furthermore, induction of the metallothionein A gene indicated that Ag had been internalized in the gills, whereas a concentration-dependant inhibition of Na/K ATPase expression indicated impaired osmoregulation at as low as 20 µg/L concentrations of Ag-NP. The commercial Ag-NP suspension caused acute gill lamellae necrosis at high concentrations (100 µg/L), potentially giving rise to the substantial (73%) fish mortality at this concentration. The two different Ag-NP preparations gave comparable results for several endpoints measured, but differed in MT-A induction and mortality, thus emphasising the variation in effects that may arise from different Ag-NP preparations.

Produced water extracts from North Sea oil production platforms result in cellular oxidative stress in a rainbow trout in vitro bioassay.

Produced water (PW) discharged from offshore oil industry contains chemicals known to contribute to different mechanisms of toxicity. The present study aimed to investigate oxidative stress and cytotoxicity in rainbow trout primary hepatocytes exposed to the water soluble and particulate organic fraction of PW from 10 different North Sea oil production platforms. The PW fractions caused a concentration-dependent increase in reactive oxygen species (ROS) after 1h exposure, as well as changes in levels of total glutathione (tGSH) and cytotoxicity after 96 h. Interestingly, the water soluble organic compounds of PW were major contributors to oxidative stress and cytotoxicity, and effects was not correlated to the content of total oil in PW. Bioassay effects were only observed at high PW concentrations (3-fold concentrated), indicating that bioaccumulation needs to occur to cause similar short term toxic effects in wild fish.

Oxidative stress responses in rainbow trout (Oncorhynchus mykiss) hepatocytes exposed to pro-oxidants and a complex environmental sample.

A wide range of pollutants in the aquatic environment have the capacity to induce toxic effects expressed as cellular oxidative stress. In the current study, the potential of an in vitro toxicity testing system was therefore investigated using rainbow trout (Oncorhynchus mykiss) hepatocytes to assess different endpoints of oxidative stress. The pro-oxidants CuSO(4) and paraquat were used as models for comparison to a complex environmental sample. Results following 6, 24, 48 and 96h exposure to different concentrations of these substances show cellular effects on intracellular ROS formation, glutathione levels and redox status, expression of the antioxidant enzymes superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase, gamma-glutamyl-cysteine synthetase (GCS) and thioredoxin, as well as cytotoxicity parameters. The most consistent effects (maximum values within brackets), observed in dose and time parameters for both model compounds and environmental sample, were the depletion of total glutathione (9.4% of control), induced levels of oxidized glutathione (695% of control), and gene expression regulation depicted relative to the control gene beta-actin of GCS mRNA (239% of control) and catalase (29% of control). In conclusion, the responses on several antioxidant defence system parameters demonstrated the validity of the in vitro toxicity testing system. Not only could multiple effects be detected at sub-lethal exposure concentrations, but these effects also gave valuable insight to the toxic mechanisms at the molecular level.

Effect-directed identification of naphthenic acids as important in vitro xeno-estrogens and anti-androgens in North sea offshore produced water discharges.

Produced water from offshore oil production platforms represents the largest direct discharge of effluent into the offshore environment. Produced water effluents contain a complex mixture of substances which are known to bind to the estrogen receptor (ER) and antagonize the androgen receptor (AR). Short-chain petrogenic alkylphenols have been identified as responsible for around 35% of the ER agonist activity measured in vitro while the compounds responsible for antagonizing the androgen receptor are unknown. For the first time we report that petrogenic naphthenic acids are weak ER agonists that account for much of the 65% of the "unknown" ER agonist potency in North Sea produced waters while also disrupting the binding of AR agonists to the AR ligand receptor. We also report other known petrogenic components such as polycyclic aromatic hydrocarbons (PAHs) and alkylphenols as environmental AR antagonists. Our investigation shows that these petrogenic components are responsible for the majority of the ER and AR receptor mediated activity in produced waters. This hypothesis is supported by data from an effects-directed analysis of produced water using normal-phase high-performance liquid chromatography (HPLC) fractionation in combination with the yeast estrogen and androgen assays as well as androgen receptor binding assays of commercially available mixtures of naphthenic acids.

Cytotoxicity of alkylphenols and alkylated non-phenolics in a primary culture of rainbow trout (Onchorhynchus mykiss) hepatocytes.

Alkylphenols are common aquatic pollutants originating from industrial use of the compounds themselves or as biodegradation products of alkylphenol polyethoxylates. The cytotoxicity of a range of alkylphenols and alkylated non-phenolics were assessed in a primary culture of rainbow trout (Onchorhynchus mykiss) hepatocytes to construct a structure-toxicity relationship for this group of ubiquitous aquatic pollutants. Metabolic inhibition and loss of membrane integrity were used as cytotoxic endpoints through use of the cellular markers Alamar blue and 5-carboxyfluorescein diacetate acetoxymethyl ester, respectively. The results show that cytotoxicity increased with the hydrophobicity of the alkylphenols for compounds with logK(OW)<4.9. Normal chained alkylphenols, branched alkylphenols and multi-substituted alkylphenols with logK(OW)4.9 deviated clearly from this relationship. The alkylphenols displayed greater cytotoxicity than alkylated non-phenolics and it is proposed that most alkylated non-phenolic caused non-polar narcosis (baseline toxicity) whereas the alkylphenols caused polar narcosis. Observations that metabolic inhibition occurred at lower concentrations than loss of membrane integrity for most chemicals indicated that interference with cellular metabolic functions was the main cause of cytotoxicity. Metabolic inhibition corresponded better than loss of membrane integrity to reported acute toxicity to fish, although the in vivo acute toxicity of hydrophobic compounds (logK(OW)>2-3) was clearly underestimated by both endpoints.

Binding of alkylphenols and alkylated non-phenolics to the rainbow trout (Oncorhynchus mykiss) plasma sex steroid-binding protein.

Alkylphenols are well-known endocrine disrupters, mediating effects through the estrogen receptor (ER). Although the estrogenic properties of the alkylphenols are well documented, alternative mechanisms of action are poorly described. In the present work, the interaction of a range of alkyl-substituted phenols and alkyl-substituted non-phenolics with the rainbow trout (Oncorhynchus mykiss) sex steroid-binding protein (rtSBP) were determined by competitive ligand-binding studies. The role of alkyl chain length and branching, substituent position, number of alkylated groups, and the requirement of a phenolic ring structure were assessed. The results showed that the rtSBP binds to most chemical structures tested, although the highest affinity was obtained for mono-substituted alkylphenols with a chain length of four to eight methyl groups. Interestingly, rtSBP binding was also observed for non-phenolic compounds such as 4-t-butylcyclohexanol and 4-t-butylnitrobenzene suggesting that the rtSBP has a broad binding specificity for alkylphenols and alkylated non-phenolics.

Effects of hypo- and hyperoxia on transcription levels of five stress genes and the glutathione system in liver of Atlantic cod Gadus morhua.

The transcript levels of three genes coding for antioxidants, Cu/Zn superoxide dismutase (SOD), catalase and phospholipid hydroperoxide glutathione peroxidase (GSH-Px), and those of two stress proteins, metallothionein (MT) and CYP1A, were examined with real-time quantitative (q) RT-PCR in hepatic tissue of Atlantic cod exposed to 46% (hypoxia), 76% (normoxia) and 145% (hyperoxia) O(2) saturation (tank outlet). To evaluate the oxidative stress state, the levels of total glutathione (tGSH), reduced glutathione (GSH) and oxidized glutathione (GSSG) and subsequently the oxidative stress index (OSI), were determined in the same tissue samples. The transcript level of GSH-Px was significantly upregulated in fish exposed to hyperoxia, and significantly downregulated in fish exposed to hypoxia, compared to the normoxia group. Significant downregulation was also found for SOD and CYP1A transcriptional levels in fish exposed to hypoxia. The transcript levels of catalase and MT did not change in liver of cod exposed to suboptimal oxygen levels. No significant differences were seen between the groups for tGSH, GSH, GSSG or OSI. Prolonged exposure to unfavourable oxygen saturation levels did not alter the OSI, indicating that the antioxidant glutathione system is maintained at an unchanged level in liver of the examined cod.

mRNA expression of antioxidant enzymes (SOD, CAT and GSH-Px) and lipid peroxidative stress in liver of Atlantic salmon (Salmo salar) exposed to hyperoxic water during smoltification.

The mRNA levels of three antioxidant genes, Cu/Zn superoxide dismutase (SOD), catalase (CAT) and phospholipid hydroperoxide glutathione peroxidase (GSH-Px), were quantified with real-time qRT-PCR in liver of Atlantic salmon Salmo salar exposed to 80% (normoxia), 105% and 130% O2 saturation for 54 days. The salmon were then translocated and exposed to 90% and 130% O2 saturation for additional 72 days during smoltification. TBARS and vitamin E levels in liver and the levels of oxidized glutathione (GSSG), total glutathione (GSH) and the resulting oxidative stress index (OSI) in blood were quantified as traditional oxidative stress markers. No significant mean normalized expression (MNE) differences of SOD, CAT or GSH-Px were found in liver after hyperoxia exposure at the two sampling times. Significantly decreased OSI was found in smolt exposed to 130% O2 saturation after 126 days (n = 18, P < 0.0001), indicating hyperoxia-induced oxidative stress. No effects were seen on growth, or on the levels of thiobarbituric reactive substances (TBARS) and vitamin E in liver after the exposure experiment. Overall, the mRNA expression of SOD, CAT and GSH-Px in liver related poorly with the hyperoxic exposure regimes, and more knowledge are needed before the expressed levels of these antioxidant genes can be applied as biomarkers of hyperoxia in Atlantic salmon.

Partial characterization of a sex steroid-binding protein in plasma from arctic charr (Salvelinus alpinus L.).

A sex steroid-binding protein (SBP) that binds 17beta-estradiol with high affinity and moderate capacity was identified in the plasma from Arctic charr (Salvelinus alpinus L.) sampled during the early stage of gonadal maturation in June and prior to spawning in October. Maximum specific binding (B(max)) and equilibrium dissociation constant (K(d)) of males (B(max) = 2122 fmol E(2)/mg protein, K(d) = 1.9 nM), females (B(max) = 4115 fmol E(2)/mg protein, K(d) = 3.0 nM), and juveniles (B(max) = 4355 fmol E(2)/mg protein, K(d) = 1.8 nM) resembled binding characteristics of SBP from related species. The early-maturing females displayed both B(max) and K(d) values significantly higher than those of males (June samples). No significant differences in binding characteristics between fully matured males or females and immature juveniles were observed in the October samples. Interestingly, despite large individual variations there was a strong correlation between SBP levels and affinity. The association rate for 17beta-estradiol was rapid (t(1/2) approximately 1-2 min) compared with the dissociation rate (t(1/2) approximately 3 h). Several native hormones (estrogens, androgens, and progesterone) were able to compete with tritiated 17beta-estradiol for the binding site. Gel filtration chromatography demonstrated a peak of estradiol binding at approximately 60 kDa, when eluted on a Sephadex S-200 HR column.

Estrogen mimics bind with similar affinity and specificity to the hepatic estrogen receptor in Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss).

A variety of organic chemicals have been documented to bind to and activate the estrogen receptor (ER) and consequently induce estrogenic effects in different animals. Although the function of the ER seems phylogenetically conserved in vertebrates, a considerable interspecies variation in the structure of the ER has been demonstrated. In this study, the hepatic ER from Atlantic salmon (Salmo salar) and rainbow trout (Oncorhunchus mykiss) were partly characterized, and the ligand-binding preference for a range of endogenous steroids and environmental estrogens (estrogen mimics) was determined by receptor-radio ligand studies. The results show that both Atlantic salmon and rainbow trout livers contain ERs that bind 1,2,4,6,7-[(3)H]estradiol ([(3)H]-E2) with high affinity and low capacity (K(d) = 2.5-4.4 nM and B(max) = 27-97 fmol/mg protein). The Atlantic salmon ER (asER) and rainbow trout ER (rtER) exhibit similar [(3)H]-E2 binding characteristics, although livers from female fish contained a two to three times higher amounts of ER than the males in the two species. In competition studies with [(3)H]-E2, the asER and rtER were found to bind both native steroids (E2 > estrone > 17 beta-estradiol 17-glucuronide >> testosterone and 11-ketotestosterone) and putative estrogen mimics (diethylstilbestrol, 4-hydroxytamoxifen, ethynylestradiol > genistein, zearalenone > 4-t-octylphenol, 4-n-nonylphenol, and o,p'-DDT). The pesticides toxaphen and dieldrin, which are proposed to bind to and activate the human ER, did not display significant binding affinity for the fish ER, however. In general, the asER and rtER were found to bind both native steroids and estrogen mimics with similar affinity and specificity. The present results suggest that closely related species such as Atlantic salmon and rainbow trout display similar ER ligand-binding requirements, although interspecies differences in ER affinity and specificity between divergent species such as fish and humans may exist.

Induction of vitellogenin synthesis in an Atlantic salmon (Salmo salar) hepatocyte culture: a sensitive in vitro bioassay for the oestrogenic and anti-oestrogenic activity of chemicals.

A variety of organic compounds have been documented to bind to the oestrogen receptor and induce oestrogenic effects in different vertebrates. The presence of these environmental oestrogens or oestrogen mimics in the aquatic environment has been suspected of disrupting the normal endocrinology of wild populations of fish. In this study, induction of vitellogenin synthesis in primary hepatocytes from Atlantic salmon (Salmo salar) was optimized and validated as an oestrogenic in vitro bioassay using a sensitive capture vitellogenin enzyme-linked immunosorbent assay. After proper optimization (cell media supplements, cell density, temperature and exposure time), this assay gave a sensitive and reproducible response to both endogenous steroids (relative potency: 17beta-oestradiol>>oestriol>oestrone>17alpha-oestradiol) and a range of common oestrogen mimics (relative potency: ethynyloestradiol and diethylstilboestrol>>genistein and zearalenone>>bisphenol A and 4-t-octylphenol>4-n-nonylphenol and 2'-chloro,4-chloro-diphenyltrichloroethane (o,p'-DDT). However, the androgen testosterone and the putative oestrogen mimics dieldrin and toxaphene were not shown to be oestrogenic using this hepatocyte bioassay. Oestrogen-induced vitellogenin synthesis was efficiently inhibited by the anti-oestrogen ZM 189.154, suggesting that this bioassay may be used for testing both the oestrogenic and the anti-oestrogenic properties of chemicals.