Aquatic toxicology (Amsterdam, Netherlands)

Published by Elsevier
Online ISSN: 0166-445X
Publications
Article
Higher water temperatures due to climate change combined with eutrophication of inland waters promote cyanobacterial blooms. Some of the cyanobacteria produce toxins leading to drinking water contamination and fish poisoning on a global scale. Here, we focused on the molecular effects of the cyanobacterial oligopeptide cyanopeptolin CP1020, produced by Microcystis and Planktothrix strains, by means of whole-genome transcriptomics. Exposure of 72hpf old zebrafish embryos for 96h to 100 and 1000μg/L CP1020 resulted in differential transcriptional alteration of 396 and 490 transcripts (fold change ≥2), respectively, of which 68 gene transcripts were common. These belong to genes related to various important biological and physiological pathways. Most clearly affected were pathways related to DNA damage recognition and repair, circadian rhythm and response to light. Validation by RT-qPCR showed dose-dependent transcriptional alterations of genes belonging to DNA damage and repair and regulation of circadian rhythm. This leads to the hypothesis that CP1020 acts on DNA and has neurotoxic activity. This transcriptome analysis leads to the identification of novel and unknown molecular effects of this cyanobacterial toxin, including neurotoxicity, which may have important consequences for humans consuming contaminated drinking water.
 
Article
Thyroid hormones (THs) play an important role in the development and metabolism of fish through their influences on genetic transcription and are targets for endocrine disruptive agents in the aquatic environment. Amitrole is a pesticide potentially interfering with thyroid hormone regulation. In this study, the rare minnow (Gobiocypris rarus) was exposed to different levels of 3-amino-1,2,4-triazole (amitrole) and allowed to recover in clean water. Plasma TH levels and the expression of TH-related genes, including transthyretin (ttr), deiodinases (d1 and d2), and the thyroid hormone receptor (tralpha) from the livers and brains were evaluated. After exposure, the plasma TH levels did not change. Histopathological observations showed that livers were degenerated at 10,000 ng/l and these damages could be recovered by the withdrawal of amitrole. However, the ttr, d1, and d2 mRNA levels in the livers of males were significantly up-regulated in all exposure groups (p<0.05). The ttr and d2 mRNA levels were significantly up-regulated at 10,000 ng/l and 10, 100, and 1000 ng/l in the livers of females, respectively (p<0.05). In the brains of males, a twofold increase of d2 mRNA levels at > or = 100 ng/l and a fivefold decrease of tralpha mRNA levels at > or = 10 ng/l were observed (p<0.05), whereas no significant differences were observed in the expression of d2 and tralpha in the brains of females. After a recovery period, the ttr, d1, and d2 mRNA levels in the livers of males returned to control levels, but the tralpha mRNA levels were irreversibly decreased at all treatments (p<0.05). In addition, the d2 mRNA levels in the livers of females were significantly induced at > or = 100 ng/l. Moreover, the d2 mRNA levels in the brains of males and females were up-regulated at 10,000 ng/l. These results indicated that amitrole exposure could result in alternations of ttr, d1, d2, and tralpha gene expression in different tissues of the rare minnow. The expression of these TH-related genes in males was more sensitive to amitrole than those of females. Recovery in clean water was associated with the selective regulation of TH-related gene transcription in the rare minnow. Therefore, these TH-related genes can serve as biomarkers to screen the effects of thyroid disruption chemicals in rare minnow.
 
Article
Tris(1,3-dichloro-2-propyl) phosphate (TDCPP) and triphenyl phosphate (TPP) are frequently detected in biota, including fish. However, knowledge of the toxicological and molecular effects of these currently used flame retardants is limited. In the present study, an in vivo screening approach was developed to evaluate effects of TDCPP and TPP on developmental endpoints and receptor-associated expression of mRNA in zebrafish embryos/larvae. Exposure to TDCPP or TPP resulted in significantly smaller rates of hatching and survival, in dose- and time-dependent manners. The median lethal concentration (LC(50)) was 7.0mg/L for TDCPP and 29.6mg/L for TPP at 120 hour post-fertilization (hpf). Real-time PCR revealed alterations in expression of mRNAs involved in aryl hydrocarbon receptors (AhRs)-, peroxisome proliferator-activated receptor alpha (PPARα)-, estrogenic receptors (ERs)-, thyroid hormone receptor alpha (TRα)-, glucocorticoid receptor (GR)-, and mineralocorticoid receptor (MR)-centered gene networks. Exposure to positive control chemicals significantly altered abundances of mRNA in corresponding receptor-centered gene networks, a result that suggests that it is feasible to use zebrafish embryos/larvae to evaluate effects of chemicals on mRNA expression in these gene networks. Exposure to TDCPP altered transcriptional profiles in all six receptor-centered gene networks, thus exerting multiple toxic effects. TPP was easily metabolized and its potency to change expression of mRNA involved in receptor-centered gene networks was weaker than that of TDCPP. The PPARα- and TRα-centered gene networks might be the primary pathways affected by TPP. Taken together, these results demonstrated that TDCPP and TPP could alter mRNA expression of genes involved in the six receptor-centered gene networks in zebrafish embryos/larvae, and TDCPP seemed to have higher potency in changing the mRNA expression of these genes.
 
Article
Measurable quantities of the selective serotonin reuptake inhibitor (SSRI), fluoxetine, have been found in surface waters and more recently in the tissues of fish. This highly prescribed pharmaceutical inhibits the reuptake of the monoamine, serotonin (5-HT; 5-hydroxytryptamine), causing a local amplification of 5-HT concentrations. Serotonin is involved in the regulation of many physiological processes in teleost fish including branchial nitrogen excretion and intestinal osmoregulation. Since the gill and intestine are directly exposed to the environment, environmental exposure to fluoxetine has the potential of affecting both these mechanisms. In the present study, we test the potential sensitivity of these processes to fluoxetine by implanting gulf toadfish, Opsanus beta, intraperitoneally with different concentrations of fluoxetine (0 (control), 25, 50, 75 and 100 microgg(-1). Fluoxetine treatments of 25 and 50 microgg(-1) were sublethal and were used in subsequent experiments. Fish treated with both 25 and 50 microgg(-1) fluoxetine had significantly higher circulating levels of 5-HT than control fish, suggesting that any 5-HT sensitive physiological process could potentially be affected by these two fluoxetine doses. However, only fish treated with 25 microgg(-1) fluoxetine showed a significant increase in urea excretion. A similar increase was not measured in fish treated with 50 microgg(-1) fluoxetine, likely because of their high circulating levels of cortisol which inhibits urea excretion in toadfish. Intestinal fluid absorption appeared to be stimulated in fish treated with 25g microgg(-1) fluoxetine but inhibited in 50 microgg(-1) treated fish. Despite these differing responses, both doses of fluoxetine resulted in lowered plasma osmolality values, which was expected based on the stimulation of fluid absorption in the 25 microgg(-1) fluoxetine-treated fish but is surprising with the 50 microgg(-1) treated fish. In the case of the latter, the corresponding stress response invoked by this level of fluoxetine may have resulted in an additional osmoregulatory response which accounts for the lowered plasma osmolality. Our findings suggest that branchial urea excretion and intestinal osmoregulation are responsive to the SSRI, fluoxetine, and further investigation is needed to determine the sensitivity of these processes to chronic waterborne fluoxetine contamination.
 
Article
The molecular mechanisms underlying nickel (Ni) and cadmium (Cd) toxicity and their specific effects on fish are poorly understood. Documenting gene transcription profiles offers a powerful approach toward identifying the molecular mechanisms affected by these metals and to discover biomarkers of their toxicity. However, confounding environmental factors can complicate the interpretation of the results and the detection of biomarkers for fish captured in their natural environment. In the present study, a 1000 candidate-gene microarray, developed from a previous RNA-seq study on a subset of individual fish from contrasting level of metal contamination, was used to investigate the transcriptional response to metal (Ni and Cd) and non metal (temperature, oxygen, and diet) stressors in yellow perch (Perca flavescens). Specifically, we aimed at (1) identifying transcriptional signatures specific to Ni and Cd exposure, (2) investigating the mechanisms of their toxicity, and (3) developing a predictive tool to identify the sublethal effects of Ni and Cd contaminants in fish sampled from natural environments. A total of 475 genes displayed significantly different transcription levels when temperature varied while 287 and 176 genes were differentially transcribed at different concentrations of Ni and Cd, respectively. These metals were found to mainly affect the transcription level of genes involved in iron metabolism, transcriptional and translational processes, vitamin metabolism, blood coagulation, and calcium transport. In addition, a linear discriminant analysis (LDA) made using gene transcription levels yielded 94% correctly reassigned samples regarding their level of metal contamination, which indicates the potential of the microarray to detect perch response to Cd or Ni effects.
 
Article
The aquatic microcosm study by Bjergager et al. (2011) on a mixture of the fungicide prochloraz and the insecticide esfenvalerate concluded that synergistic effects were found at environmentally realistic concentrations for these compounds and thus that current risk assessment procedures might underestimate the effects of synergistically interacting azoles and pyrethroids. Both prochloraz and esfenvalerate are registered in Europe and thus the relevance of the employed concentrations can be assessed against European surface water measurements and risk assessments procedures. A detailed comparison of the employed concentration of prochloraz in the microcosm study with the concentration deemed acceptable in the European Union and those actually measured in the aquatic environment demonstrate that the employed prochloraz concentration was about two orders of magnitude too high. Therefore, on basis of the data presented by Bjergager et al. (2011) it cannot be concluded that current European single substance risk assessment procedures are insufficiently protective and that synergism actually occurs at environmentally relevant concentrations.
 
Article
Cyanobacteria are prokaryotic algae found in oceans and freshwaters worldwide. These organisms are important primary producers in aquatic ecosystems because they can provide essential food for grazers and herbivores. In this study, the physiological and biochemical responses of the freshwater cyanobacterium Synechococcus sp. PCC7942 to two organic booster biocides Irgarol 1051 and diuron were compared and evaluated using 96 h growth tests in a batch-culture system. The 96 h median effective concentrations (EC(50)) were 0.019 and 0.097 μmol L(-1) for Irgarol 1051 and diuron, respectively, which indicate that Irgarol 1051 is about 5 times more toxic than diuron to cyanobacteria. Moreover, remarkable physiological and biochemical responses occurred in the Irgarol 1051 and diuron treatments. Irgarol 1051 and diuron stimulated cyanobacterial growth, increased the soluble protein content, and enhanced the catalase (CAT) activity at low concentrations, but inhibited them at high concentrations. However, the malondialdehyde (MDA) and polysaccharide content of the cyanobacteria were only significantly affected by Irgarol 1051. These observations suggest that Irgarol 1051 and diuron are toxic to Synechococcus sp. PCC7942, and their use should be restricted in maritime industries.
 
Article
The herbicides Irgarol 1051 (2-(tert-butylamino)-4-cyclopropylamino)-6-(methylthio)-1,3,5-triazine) and Diuron (3-(3',4'-dichlorophenyl)-1,1-dimethylurea) are commonly incorporated into antifouling paints to boost the efficacy of the compound towards algae. Previous investigations have identified environmental concentrations of these herbicides as being a threat to non-target organisms, such as seagrasses. Their individual toxicity has been assessed, but they can co-occur and interact, potentially increasing their toxicity and the threat posed to seagrass meadows. Chlorophyll fluorescence (Fv:Fm) and leaf specific biomass ratio (representing plant growth) were examined in Zostera marina L. after a 10-day exposure to the individual herbicides. The EC20 for each herbicide was determined and these then used in herbicide mixtures to assess their interactive effects. Irgarol 1051 was found to be more toxic than Diuron with lowest observable effect concentrations for Fv:Fm reduction of 0.5 and 1.0 +/- microg/l and 10-day EC50 values of 1.1 and 3.2 microg/l, respectively. Plants exposed to Irgarol 1051 and Diuron showed a significant reduction in growth at concentrations of 1.0 and 5.0 microg/l, respectively. When Z. marina was exposed to mixtures, the herbicides commonly interacted additively or antagonistically, and no significant further reduction in photosynthetic efficiency was found at any concentration when compared to plants exposed to the individual herbicides. However, on addition of the Diuron EC20 to varying Irgarol 1051 concentrations and the Irgarol 1051 EC20 to varying Diuron concentrations, significant reductions in Fv:Fm were noted at an earlier stage. The growth of plants exposed to Diuron plus the Irgarol 1051 EC20 were significantly reduced when compared to plants exposed to Diuron alone, but only at the lower concentrations. Growth of plants exposed to Irgarol 1051 and the Diuron EC20 showed no significant reduction when compared to the growth of plants exposed to Irgarol 1051 alone. Despite the addition of the EC20 not eliciting a further significant reduction when compared to the herbicides acting alone for most of the mixtures, the lowest observable significant effect concentration for growth and photosynthetic efficiency decreased to 0.5 microg/l for both herbicides. Irgarol 1051 and Diuron have been shown to occur together in concentrations above 0.5 microg/l, suggesting that seagrasses may be experiencing reduced photosynthetic efficiency and growth as a result.
 
Article
Alkylphenols including 4-tert-pentylphenol (4-PP) have been shown to alter sexual differentiation in fish due to their estrogenic properties. Medaka (Oryzias latipes) is so sensitive to these substances because morphological sex reversal and testis-ova induction are well developed in the exposed males. However, little work has been done to characterize the molecular effects of estrogenic substances on the morphological and gonadal feminization in male fish. Cytochrome P450 11beta-hydroxylase (P450(11beta)) is a key steroidogenic enzyme in production of 11-ketotestosterone which is the predominant androgen in male fish. In this study, we cloned a cDNA encoding medaka testicular P450(11beta), and then investigated the gene expression of P450(11beta) in the testes of genetically male medaka exposed to 4-PP. The cDNA contains 1740 nucleotides that encode a protein of 543 amino acids, which shares 68.9% and 73.4% homology with testicular P450(11beta)s from Japanese eel (Anguilla japonica) and rainbow trout (Oncorhynchus mykiss), respectively. HeLa cells transfected with an expression vector containing the open reading frame of medaka P450(11beta) cDNA showed 11beta-hydroxylating activity in the presence of exogenous testosterone. Analysis of tissue distribution by RT-PCR showed great abundance of P450(11beta) mRNA in testis. In the partial life-cycle exposure with 4-PP, morphologically sex-reversal was observed in XY medaka exposed to 4-PP concentrations of > or =238 microg/L. Furthermore, exposure to 4-PP completely inhibited P450(11beta) mRNA expression in the gonads of sex-reversed XY fish at 60-day posthatch. These results suggest that xeno-estrogen 4-PP may have inhibitory effects on the synthesis of testicular 11-oxygenated androgens through downregulation of P450(11beta) expression in the genetically male fish.
 
Article
The brown bullhead Ameiurus nebulosus is a species of the family Ictaluridae commonly used as a sentinel of environmental contamination. While these fish have been utilized for this purpose in areas contaminated with polychlorinated biphenyls (PCBs), few controlled, laboratory-based studies have been designed to document the effects of PCB mixtures in this species. Here, brown bullhead were exposed to the PCB mixture, Aroclor 1248, via intraperitoneal injection and the effects on immune function, plasma hormones and disease resistance were evaluated. Exposure to this mixture led to a decrease in bactericidal activity and circulating antibodies to Edwardsiella ictaluri present from a previous exposure to this pathogen. A subsequent E. ictaluri disease challenge led to significantly higher mortality in A1248 treated fish compared to vehicle-control fish. The mitogenic response to the T-cell mitogen, phytohemaglutinin-P, was increased compared to vehicle-control fish. The steroid hormone, cortisol, and the thyroid hormone, T3, were also significantly lower in A1248 exposed fish. In summary, we have validated a number of functional immune assays for application in brown bullhead immunotoxicity studies. Additionally, we have demonstrated that the PCB mixture (A1248) modulates both immune function and endocrine physiology in brown bullhead. Such data may compliment the interpretation of data yielded from applied field studies conducted in PCB contaminated aquatic ecosystems.
 
Article
The sensitivity of carp to chronical stress due to single and combined mixtures (each 50%) of TBT and PCB was evaluated under laboratory conditions. And concluded, mixed exposures were partially more or less toxic than single ones. Xenobiotic stress led to both decreased mean daily swimming speed and an increased mean swimming speed during darkness. Also a decreased preferred swimming depth (nearer to the surface) during nighttime was observed. We found approximately synergistic effects of TBT and PCB in swimming behavior of carp exposed to those mixtures. PCB did not affect the body growth significantly, TBT-stress led to a decreased body growth and the exposure to PCB-TBT mixture caused an approximately additive decrease of body growth. Measurement of biotransformation potential measured as GST enzyme activity showed both increasing or decreasing activity levels after exposure to single and mixture chemical combinations (approximately additive, antagonistic and synergistic). Nevertheless, we had to conclude that all methods tested were useful to screen subacute effects of single as well as mixed xenobiotic chemicals on carp, which is the prerequisite for an investigation of samples taken from the environment.
 
Article
Polychlorinated biphenyls (PCBs) are a widespread aquatic contaminant and are present in both wild and hatchery raised Atlantic salmon, Salmo salar. The possible sub-lethal alterations in smolt physiology and behavior due to PCB exposure of salmon have not been widely examined. In this study, we examined the effects of the PCB mixture Aroclor 1254 on survival and smolt development of Atlantic salmon. In separate experiments, fish were exposed as yolk-sac larvae or as juveniles just prior to the parr-smolt transformation in April to 1 microgl(-1) (PCB-1) or 10 microgl(-1) (PCB-10) aqueous Aroclor 1254 (A1254), or vehicle for 21 days. After exposure, yolk-sac larvae were reared at ambient conditions for 1 year, until the peak of smolting the following May. Juveniles were sampled immediately after exposure. Both groups were assessed for behavioral, osmoregulatory, and endocrine disruption of smolt development at the peak of smolting. PCB-1 and PCB-10 treated yolk-sac larvae exhibited significant increases in the rate of opercular movement after 14 and 21 days of exposure. At the peak of smolting, prior exposure as yolk-sac larvae to PCB-1 did not affect behavior, while PCB-10 dramatically decreased volitional preference for seawater. Neither concentration of A1254 had long-term effects on the osmoregulatory or endocrine parameters measured in animals exposed as yolk-sac larvae. Juvenile fish exposed to PCB-1 or PCB-10 during smolting exhibited a dose-dependent reduction in preference for seawater. Fish treated with the higher dose of A1254 also exhibited a 50% decrease in gill Na(+),K(+)-ATPase activity and a 10% decrease in plasma chloride levels in freshwater. In addition, plasma triiodothyronine was reduced 35-50% and plasma cortisol 58% in response to exposure to either concentration; whereas plasma thyroxine, growth hormone, and insulin-like growth factor I levels were unaffected. These results indicate that the effects of exposure to A1254 may vary according to developmental stage. Exposure to A1254 in the freshwater environment can inhibit preparatory adaptations that occur during smolting, thereby reducing marine survival and sustainability of salmon populations.
 
Article
Polychlorinated biphenyls (PCBs) exist as persistent organic pollutants in numerous river systems in the United States. Unfortunately, some of these rivers are sites of active Atlantic salmon restoration programs, and polychlorinated biphenyls have been implicated as ancillary factors contributing to failed salmon restoration. Here, we investigate the immediate and chronic effects of intermediate duration aqueous PCB exposure (1 or 10 microgL-1 Aroclor 1254) on the mitogen-stimulated lymphoproliferative response of Atlantic salmon anterior kidney leukocytes (AKLs). A short-term study was designed to examine immunomodulation in Atlantic salmon smolts immediately following 21 days of aqueous exposure, while a long-term study evaluated chronic impacts in the mitogen response in parr 15 months post-exposure as larvae. The proliferative response of AKLs to the mitogens concanavalin A (CON A), phytohemaglutinnin-P (PHA-P), pokeweed mitogen (PWM), and lipopolysaccharide were used as an indice of immunomodulation. The proliferative response to the T-cell mitogens CON A and PHA-P was significantly increased in the 10 microgL-1 group (n=10; P=0.043 and 0.002, respectively) immediately following exposure of smolts. Additionally, The PHA-P response was significantly increased in the 1 microgL-1 exposure group (n=10, P=0.036). In fish treated as larvae and tested 15 months later, the PHA-P sensitive populations exhibited elevated proliferation in the 1 and 10 microgL-1 groups (n=12, P<0.04) relative to the vehicle control while the PWM response was significantly increased (n=12, P=0.036) only in the 10 microgL-1 treated groups. These results demonstrate an immunomodulatory effect of PCBs on T-cell mitogen sensitive populations of lymphocytes in Atlantic salmon as well as long-term immunomodulation in PHA-P and PWM sensitive populations.
 
Article
Exposure to dioxin-like chemicals that activate the aryl hydrocarbon receptor (AHR) can result in increased cellular and tissue production of reactive oxygen species (ROS). Little is known of these effects during early fish development. We used the fish model, Fundulus heteroclitus, to determine if the AHR ligand and pro-oxidant 3,3',4,4',5-pentachlorobiphenyl (PCB126) can increase ROS production during killifish development, and to test a novel method for measuring ROS non-invasively in a living organism. The superoxide-sensitive fluorescent dye, dihydroethidium (DHE), was used to detect in ovo ROS production microscopically in developing killifish exposed to PCB126 or vehicle. Both in ovo CYP1A activity (ethoxyresorufin-o-deethylase, EROD) and in ovo ROS were induced by PCB126. In ovo CYP1A activity was inducible by PCB126 concentrations as low as 0.003 nM, with maximal induction occurring at 0.3 nM PCB126. These PCB126 concentrations also significantly increased in ovo ROS production in embryonic liver, ROS being detectable as early as 5 days post-fertilization. These data demonstrate that the pro-oxidant and CYP1A inducer, PCB126, increases both CYP1A activity and ROS production in developing killifish embryos. The superoxide detection assay (SoDA) described in this paper provides a semi-quantitative, easily measured, early indicator of altered ROS production that can be used in conjunction with simultaneous in ovo measurements of CYP1A activity and embryo development to explore functional relationships among biochemical, physiological and developmental responses to AHR ligands.
 
Article
The endocrine system of wildlife is exposed to a wide variety of natural and man-made chemicals which may lead to damage to the reproductive system and other adverse effects, including alteration of drug-metabolizing enzymes. In the present study, the effects of in vivo exposure to a natural (17beta-estradiol: E2) or a xenoestrogen (4-nonylphenol: NP) estrogen or an anti-estrogen (3,3',4,4',5-pentachlorobiphenyl: PCB 126) upon vitellogenin (VTG) synthesis and hepatic phase 1 and 2 enzymes have been investigated in adult male sea bass. By means of ELISA analysis with the use of polyclonal antibodies prepared against VTG purified from E2-treated sea bass, we assessed the time course and sensitivity of VTG induction in the plasma of sea bass treated with E2 at 0.1, 0.5, 2.5 and 5.0 mg/kg doses or NP at 5.0 or 50 mg/kg doses, respectively. Sea bass sensitivity to this induction was found to be similar to that of other fish species, but with a delay in maximal response. E2 treatment also caused a selective time- and dose-dependent inhibition of hepatic CYP1A-linked EROD and phase 2 glutathione S-transferase (GST) activities, without affecting the activity of CYP3A-linked 6beta-testosterone hydroxylase, (omega)- and (omega-1)-lauric acid hydroxylases or phase 2 DT-diaphorase. A similar selective inhibition on CYP1A was also observed in fish treated with 50 mg/kg NP. The results regarding CYP1A and CYP3A were also confirmed by Western blot analysis. When the sea bass were treated with either 10 or 100 microg/kg PCB 126, an AhR ligand not yet tested in vivo in fish to assess its anti-estrogenicity, a modest and selective induction of EROD and DT-diaphorase activities was observed. Interestingly, both these activities were recovered to their control levels in sea bass co-treated with 0.5 mg/kg E2 and 10 or 100 microg/kg PCB 126, probably through a cross-talk mechanism between the estrogen receptor and AhR or other transcription factors that regulate the expression of these enzymes. Furthermore, it was demonstrated that PCB 126 possesses a potent anti-estrogenic activity in the sea bass in vivo as it inhibited the E2-induced VTG synthesis with an IC50 of 28 microg/kg. The results of this study suggest that the exposure of fish to xenoestrogens or anti-estrogens may alter, in addition to various physiological processes, the expression of specific CYPs and phase 2 enzymes, thereby reducing the capability of their detoxication system.
 
Article
The AHR pathway activates transcription of CYP1A and mediates most toxic responses from exposure to halogenated aromatic hydrocarbon contaminants such as PCBs and PCDD/Fs. Therefore, expression of CYP1A is predictive of most higher level toxic responses from these chemicals. To date, no study had developed an assay to quantify CYP1A expression in any sturgeon species. We addressed this deficiency by partially characterizing CYP1A in Atlantic sturgeon (Acipenser oxyrinchus oxyrinchus) and shortnose sturgeon (Acipenser brevirostrum) and then used derived sturgeon sequences to develop reverse transcriptase (RT)-PCR assays to quantify CYP1A mRNA expression in TCDD and PCB126 treated early life-stages of both species. Phylogenetic analysis of CYP1A, CYP1B, CYP1C and CYP3A deduced amino acid sequences from other fishes and sturgeons revealed that our putative Atlantic sturgeon and shortnose sturgeon CYP1A sequences most closely clustered with previously derived CYP1A sequences. We then used semi-quantitative and real-time RT-PCR to measure CYP1A mRNA levels in newly hatched Atlantic sturgeon and shortnose sturgeon larvae that were exposed to graded doses of waterborne PCB126 (0.01-1000 parts per billion (ppb)) and TCDD (0.001-10 ppb). We initially observed significant induction of CYP1A mRNA compared to vehicle control at the lowest doses of PCB126 and TCDD used, 0.01 ppb and 0.001 ppb, respectively. Significant induction was observed at all doses of both chemicals although lower expression was seen at the highest doses. We also compared CYP1A expression among tissues of i.p. injected shortnose sturgeon and found significant inducibility in heart, intestine, and liver, but not in blood, gill, or pectoral fin clips. For the first time, our results indicate that young life-stages of sturgeons are sensitive to AHR ligands at environmentally relevant concentrations, however, it is yet to be determined if induction of CYP1A can be used as a biomarker in environmental biomonitoring.
 
Concentrations (whole fish, ng g − 1 wet weight) of 14 C-PCB 126 in juvenile rainbow trout exposed to dietary concentrations of 12.4 and 126 ng g − 1 of PCB 126 for 30 days followed by 160 days of non-spiked food. Each point is the mean concentration of three fish9 S.E.M.  
Bioaccumulation parameters of PCB 126 from dietary exposures using juvenile rainbow trout
Mean values for retinoid stores and tocopherol in livers of rainbow trout exposed to PCB 126 for 30 days. Histogram bars represent mean and S.E.M. Significant differences (PB0.05) between reference and exposed sites are indicated by an asterisk.  
EROD activity in livers (postmitochondrial supernatants ) relationships with whole body concentrations of PCB 126 in juvenile rainbow trout exposed to dietary concentrations of 12.4 and 126 ng g − 1 of PCB 126 for 30 days followed by 160 days of non-spiked food. Each point is the mean 9 S.E.M. of the EROD activity in three fish livers and PCB 126 concentration of three fish. Dashed lines are the liner regression of the low (closed circles, slope =2.30; r 2 = 0.76; P= 0.02) and high (open circles, slope = 2.14; r 2 = 0.85; PB 0.01) exposed fish and the solid line is the regression for all points (slope= 1.01, r 2 = 0.76, PB0.001).  
Article
Juvenile rainbow trout (Oncorhynchus mykiss) (initial weights 2-5 g) were exposed to three dietary concentrations (0, 12.4 and 126 ng g(-1), wet weight) of a 14C-labelled 3,3',4,4',5-pentachlorobiphenyl (PCB 126) for 30 days followed by 160 days of clean food. We assessed bioaccumulation, histology (liver and thyroid) and biochemical responses (liver ethoxyresorufin-O-deethylase (EROD), liver vitamins (retinoids and tocopherol) and muscle thyroid hormone levels) along with growth and survival. The half-life of PCB 126 in the rainbow trout ranged from 82 to 180 days while biomagnification factors (BMF) ranged from 2.5 to 4.1 providing further evidence that PCB 126 is among the most bioaccumulative PCB congeners. Toluene extractable 14C declined with time in the trout suggesting the possibility of some biotransformation and/or covalent bonding with biological macromolecules. The threshold for liver EROD induction by PCB 126 was approximately 0.1 ng g(-1) (wet weight). EROD activities in the low- and high treatments were 9 and 44 times greater than control, respectively, and remained elevated throughout the experiment. EROD activity was correlated with whole body concentrations of PCB 126 although there was evidence of EROD activity suppression in the highly exposed fish. Liver didehydroretinoids and tocopherol concentrations were depressed by the high PCB 126 dose after 30 days exposure. Initially, muscle concentrations of thyroxine (T4) and triiodo-L-thyronine (T3) declined as the fish grew during the experiment, and exposure to PCB 126 accelerated the growth related decline. More information is needed to assess the functional significance of the reduced muscular stores of thyroid hormones. Despite the changes in liver EROD, liver vitamins and muscle thyroid hormones, liver and thyroid histology in trout examined after 30 days exposure and growth parameters were unaffected by PCB 126. This indicates that the functional competences of the physiological factors associated with growth were maintained under the experimental conditions.
 
Article
Bone is a dynamic tissue with diverse functions including growth, structural support, pH balance and reproduction. These functions may be compromised in the presence of organopollutants that can alter bone properties. We exposed juvenile diamondback terrapins (Malaclemys terrapin) to 3,3',4,4',5-pentachlorobiphenyl (PCB 126), a ubiquitous anthropogenic organochlorine, and measured organic content, apparent bone mineral density (aBMD) using radiography and computed tomography, and quantified bone microstructure using histological preparations of femora. PCB-exposed terrapins were smaller in total size. Skulls of exposed animals had a higher organic content and a skeletal phenotype more typical of younger animals. The femora of exposed individuals had significantly reduced aBMD and significantly more cortical area occupied by non-bone. Because bone is an integral component of physiology, the observed skeletal changes can have far-reaching impacts on feeding and locomotor performance, calcium reserves and ultimately life history traits and reproductive success. Additionally, we caution that measurements of bone morphology, density, and composition from field-collected animals need to account not only for relatedness and age, but also environmental pollutants.
 
Article
Exposure of developing fish to polycyclic aromatic hydrocarbons (PAHs) and halogenated aromatic hydrocarbons (HAHs) results in a suite of defects including cardiac malformation, pericardial and yolk sac edema, craniofacial defects, and hemorrhaging. Several populations of Atlantic killifish or mummichog (Fundulus heteroclitus) on the Atlantic coast of the United States are resistant to the developmental and acute toxicity caused by PAHs and HAHs; this has made Fundulus a valuable model for studying aryl hydrocarbon sensitivity and adaptation. In order to further increase the utility of Fundulus, better understanding of the components of the molecular pathways governing aryl hydrocarbon response in Fundulus is required. The aryl hydrocarbon receptor (AHR) is known to mediate many of the toxic responses to PAHs and HAHs. A single AHR has been identified in mammals, but Fundulus has two AHRs and their relative roles are not clear. In the current study, translation-blocking and splice-junction morpholino gene knockdown was used to determine the roles of AHR1 and AHR2 in mediating cardiac teratogenesis induced by beta-naphthoflavone (BNF), benzo[k]fluoranthene (BkF), and 3,3',4,4',5-pentachlorobiphenyl (PCB-126). Here we report that AHR2 and not AHR1 knockdown resulted in rescue of teratogenicity induced by BNF, BkF, and PCB-126. These data demonstrate that AHR2 is the primary mediator of cardiac teratogenesis caused by multiple aryl hydrocarbons in Fundulus and suggest that suppression of the AHR pathway through modulation of AHR2 is a plausible mechanism for PAH resistance in adapted fish. Additionally, this is the first reported use of splice-junction morpholinos in Fundulus.
 
Article
Two experiments were performed to study the interaction between benzo[a]pyrene (BaP) and the planar, dioxin-like PCB congener 3,3',4,4',5-pentachlorobiphenyl (CB 126) in the flatfish dab (Limanda limanda). The first experiment involved four groups. Group I was treated with 10 µg/kg CB 126, group II was treated with 2 mg/kg BaP, group III was first treated with 10 µg/kg CB 126 and exposed to 2 mg/kg BaP 6 days later, and group IV was a control group. The second experiment was similar, except that the BaP dosage level was increased to 50 mg/kg. Pre-treatment with 10 µg/kg of CB 126 always caused the induction of hepatic cytochrome P450 1A (CYP1A), as measured by significant increases of the model reaction 7-ethoxyresorufin-O-deethylase (EROD) in microsomal preparations. Treatment of dab with BaP caused a significant EROD induction at the 50 mg/kg, but not at the 2 mg/kg level. Concurrent with EROD induction by either CB 126 or 50 mg/kg BaP, was a significant change in the biliary metabolite pattern in favour of 1-hydroxybenzo[a]pyrene and 3-hydroxybenzo[a]pyrene and towards a lower fraction of the procarcinogen BaP-7,8-dihydrodiol (7,8-DIOL). Pre-treatment with CB 126 did not cause an increase of hepatic BaP DNA adducts formed after treatment with either 2 or 50 mg/kg BaP. Glutathione S-transferase (GST) activities remained also unaffected by any of the treatments. The results of this study suggest that the pattern of BaP metabolites in bile depends on the level of CYP1A induction. Moreover, the concurrence of a potent CYP1A inducer and BaP does not necessarily lead to an increase in DNA adduct levels in liver tissue. The observation that the level of 7,8-DIOL is decreased despite a higher (CYP1A mediated) EROD activity explains, at least in part, the lack of induction of DNA adducts.
 
Article
Various environmental contaminants are known agonists for the aryl hydrocarbon receptor (AHR), which is highly conserved across vertebrate species. Due to gene duplication events before and after the divergence of ray- and lobe-finned fishes, many teleosts have multiple AHR isoforms. The zebrafish (Danio rerio) has three identified AHRs: AHR1A and AHR1B, the roles of which are not yet well elucidated, and AHR2, which has been shown to mediate the toxicity of various anthropogenic compounds including dioxins, polychlorinated biphenyls (PCBs), and polycyclic aromatic hydrocarbons (PAHs). In this study, we sought to explore the role of the two AHR1 isoforms in PAH- and PCB-induced toxicity in zebrafish embryos utilizing morpholino gene knockdown of the AHR isoforms. Knockdown of AHR1B did not affect the toxicity of PAH mixtures or PCB-126, whereas knockdown of AHR1A exacerbated the cardiac toxicity caused by PAH mixtures and PCB-126. Knockdown of AHR1A did not impact the mRNA expression of CYP1A, CYP1B1, and CYP1C1 in exposed embryos, but it did result in increased CYP1 activity in exposed embryos. As has been shown previously, knockdown of AHR2 resulted in protection from PAH- and PCB-induced cardiac deformities and prevented CYP1 enzyme activity in exposed embryos. Co-knockdown of AHR1A and AHR2 resulted in an intermediate response compared to knockdown of AHR1A and AHR2 individually; co-knockdown did not exacerbate nor protect from PAH-induced deformities and embryos exhibited an intermediate CYP1 enzyme activity response. In contrast, co-knockdown of AHR1A and AHR2 did protect from PCB-126-induced deformities. These results suggest that AHR1A is not a nonfunctional receptor as previously thought and may play a role in the normal physiology of zebrafish during development and/or the toxicity of environmental contaminants in early life stages.
 
Article
The branched isomers of p-nonylphenol (NP) are perceived to be more resistant to biodegradation in aquatic environments as well as to have more estrogen-like toxicity than the straight chain isomers. By use of GC-MS, some of them have been identified and found to exist in higher concentrations in the isomeric compound mixture than the straight chain isomers. The investigations of the distribution and metabolism of these branched isomers in aquatic organisms are therefore considered to be important in understanding the mechanisms of toxicity of NP. A single tertiary isomer of NP, 4(3'-,6'-dimethyl-3'-heptyl)-phenol, was synthesized in the laboratory and used in in vivo studies of its organ distribution and metabolism in Lymnaea stagnalis L., following a constant exposure of the organisms to 14C-NP isomer in water over a period of 8 days at an average exposure concentration of 105 ppb (range: 93-116 ppb). The results obtained clearly showed the distribution and bioconcentration of the isomer residues in various internal organs of Lymnaea after uptake in water and food. Analysis of the extracts of the organ tissues and faeces by HPLC and GC-MS after digestion with Pankreatin/beta-glucuronidase and nitric acid, respectively, showed that the isomer was metabolized by conjugation to glucuronic acid and hydroxylation to a catechol. The findings from these studies and their implications in the biotransformation and estrogenicity of NP in Lymnaea stagnalis L. are further discussed in detail in this paper.
 
Article
Ortho-substituted PCBs intercalate between membrane phospholipids similarly to cholesterol and increase fluidity. Ectothermic animals have a well-developed homeoviscous response to counter the fluidising effect of temperature and avoid the disruption of membrane proteins. However, it remains unknown whether chemical fluidisation can also activate a homeoviscous response or interfere with normal acclimation to temperature. The fatty acid composition and cholesterol content of membranes from gill, white muscle, liver, and brain was measured in goldfish exposed to 4 treatments in a 2×2 factorial design (acclimated to 5 or 20°C, and exposed or not to PCB-153). The expression of Δ6 and Δ9 desaturases was also measured in gill and liver because these enzymes modulate changes in membrane unsaturation. We hypothesised that thermal and chemical stress would cause similar adjustments in phospholipid unsaturation, membrane cholesterol, and desaturase expression. Results show that PCB-153 triggers a homeoviscous response by changing cholesterol content in liver (+51%) and brain (+216%), as well as the double bond index in gills (-17%). In response to higher temperature, the membranes of gill, muscle, and brain substitute polyunsaturated fatty acids such as arachidonate [20:4] and eicosadienoate [20:2] with saturated fatty acids such as palmitate [16:0] and stearate [18:0]. Each tissue has a distinct pattern of changes, suggesting that different local factors contribute to the stress response. It is also possible that the thermal tolerance of individual species influences the homeoviscous response because the changes observed in goldfish liver are not consistent with what has been reported for trout liver. No evidence supporting the activation of desaturase expression could be found. Overall, and contrary to expectation, modulating membrane cholesterol is the main mechanism used to cope with PCB-153, whereas changes in unsaturation dominate temperature acclimation. If also present in other species, these protective responses may prove particularly important for polar fish that face the combined effects of thermal stress from climate change and chemical stress from organochlorine deposition. This study is the first to show that in vivo exposure to a membrane fluidiser can cause a homeoviscous response in an ectothermic animal. We conclude that the homeostatic mechanisms that preserve normal membrane function vary: (1) with the nature of the stress that perturbs fluidity, (2) with local conditions within each tissue, and (3) possibly with the thermal tolerance of individual species. These complicating factors will have to be considered in future studies of homeoviscous adjustments.
 
Article
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.
 
Article
For a predictive assessment of the aquatic toxicity of chemical mixtures, two competing concepts are available: concentration addition and independent action. Concentration addition is generally regarded as a reasonable expectation for the joint toxicity of similarly acting substances. In the opposite case of dissimilarly acting toxicants the choice of the most appropriate concept is a controversial issue. In tests with freshwater algae we therefore studied the extreme situation of multiple exposure to chemicals with strictly different specific mechanisms of action. Concentration response analyses were performed for 16 different biocides, and for mixtures containing all 16 substances in two different concentration ratios. Observed mixture toxicity was compared with predictions, calculated from the concentration response functions of individual toxicants by alternatively applying both concepts. The assumption of independent action yielded accurate predictions, irrespective of the mixture ratio or the effect level under consideration. Moreover, results even demonstrate that dissimilarly acting chemicals can show significant joint effects, predictable by independent action, when combined in concentrations below individual NOEC values, statistically estimated to elicit insignificant individual effects of only 1%. The alternative hypothesis of concentration addition resulted in overestimation of mixture toxicity, but differences between observed and predicted effect concentrations did not exceed a factor of 3.2. This finding complies with previous studies, which indicated near concentration-additive action of mixtures of dissimilarly acting substances. Nevertheless, with the scientific objective to predict multi-component mixture toxicity with the highest possible accuracy, concentration addition obviously is no universal solution. Independent action proves to be superior where components are well known to interact specifically with different molecular target sites, and provided that reliable statistical estimates of low toxic effects of individual mixture constituents can be given. With a regulatory perspective, however, fulfilment of both conditions may be regarded as an extraordinary situation, and hence concentration addition may be defendable as a pragmatic and precautionary default assumption.
 
Article
We studied how lead is bioconcentrated and distributed in the rotifer Brachionus calyciflorus using metal histochemistry to locate lead granules, Leadmium Green® analysis to establish the route of uptake, atomic absorption to determined the bioconcentration factor (BCF), and detected the presence of microelements in the cuticle by X-ray microanalysis with scanning electron microscopy. Our results indicate: (a) the digestive system is the main route of lead uptake in the rotifer B. calyciflorus, (b) after 24-h lead is deposited in granules in the mastax and vitellarium, (c) our energy-dispersive X-ray microanalysis indicates decalcification taking place in the cuticle of the rotifer after a 24-h lead exposure, and (d) we determined a BCF = 115 for lead after a 24 h exposure. However, the route of mobilization and storage of intracellular lead are still not fully understood in B. calyciflorus.
 
Article
Cytochrome P450s (CYPs) are important xenobiotic metabolizing proteins. While their functions are well understood in mammals, CYP function in non-mammalian vertebrate systems is much less defined, with function often inferred from mammalian data, assuming similar function across vertebrate species. In this study, we investigate whether in vivo treatment with known mammalian CYP inducers can alter the in vitro catalytic activity of fish microsomes using eleven fluorescent CYP-mediated substrates. We investigate the basal metabolism and induction potential for hepatic CYPs in two fish species, rainbow trout (Oncorhynchus mykiss) and killifish (Fundulus heteroclitus). Species differences were found in the baseline metabolism of these substrates. Killifish have significantly higher metabolic rates for all tested substrates except 7-benzyloxyquinoline and 7-benzyloxy-4-trifluoromethylcoumarin (both mammalian CYP3A substrates); significant differences were also seen between male and female killifish. Treatment with dexamethasone, pregnenolone-16alpha-carbonitrile, and rifampicin did not cause broad, measurable CYP induction in either fish species. In trout, dexamethasone (100 mg kg(-1)) significantly induced 3-cyano-7-ethoxycoumarin metabolism and rifampicin (100 mg kg(-1)) induced the dealkylation of 7-methoxyresorufin, although both were highly variable. Female killifish exposed to pregnenolone-16alpha-carbonitrile (100 mg kg(-1)) showed significantly higher metabolism of 7-pentoxyresorufin. Overall, dexamethasone, pregnenolone-16alpha-carbonitrile and rifampicin did not appear to consistently increase CYP activity in fish. Trout treated with 10 or 50 mg kg(-1) beta-naphthoflavone (BNF), a CYP1A inducer, showed significantly induced activity across almost all substrates tested, exceptions being 7-benzyloxyquinoline, 7-benzyloxy-4-trifluoromethylcoumarin and dibenzylfluorescein. 7-Methoxy-4-(aminomethyl)coumarin, a typical CYP2D substrate in mammals, was not metabolized by untreated fish liver microsomes; however, treatment with BNF significantly induced the metabolism of this substrate in trout. Induced substrate metabolism in BNF-treated microsomes was only correlated across selective substrates, suggesting that BNF induces multiple CYPs in fish liver. These include the known BNF inducible CYP1s plus a number of as yet unidentified fish CYPs. Overall, many of these catalytic assays could be valuable tools for identification of the function of specific CYP subfamilies and individual isoforms in fish.
 
Article
Research was conducted to determine the kinetics of hepatic vitellogenin (VTG) mRNA regulation and plasma VTG accumulation and clearance in male sheepshead minnows (Cyprinodon variegatus) during and after cessation of exposure to either 17 beta-estradiol (E2) or para-nonylphenol (NP). Adult fish were continuously exposed to aqueous measured concentrations of 0.089 and 0.71 microg E2 per l, and 5.6 and 59.6 microg NP per l for 16 days using an intermittent flow-through dosing apparatus. Fish were sampled on days 8 and 16 of exposure followed by sampling at discrete intervals for up to 96 days post-exposure. At each interval five fish were randomly sampled from each concentration and hepatic VTG mRNA and serum VTG levels for individual fish determined by slot blot and direct enzyme-linked immunosorbent assay (ELISA), respectively. Exposure to E2 and NP resulted in a dose dependent increase in hepatic VTG mRNA and plasma VTG over the course of the 16-day exposure period. Mean plasma VTG levels at day 16 were >100 mg/ml for both high doses of E2 and NP, and >20 mg/ml for the low exposure treatments. Within 8 days post-exposure, hepatic VTG mRNA levels returned to baseline in both high and low E2 treatments but remained elevated 2-4 fold in the NP treatments. Due to a shortened sampling period, a clearance rate for plasma VTG in the 5.6 microg NP per l treatment could not determined. In the 0.089, 0.71 microg E2 per l, and 59.6 microg NP per l treatments, VTG levels began decreasing within 4 days after exposure cessation and exhibited an exponential rate of elimination from plasma. Clearance rates for 0.71 microg E2 per l and 59.6 microg NP per l were not significantly different (P=0.47), however, both demonstrated significantly higher rates of clearance (P<0.02) than observed in the 0.089 microg E2 per l treatment. Our results indicate that hepatic VTG mRNA rapidly diminishes after cessation of estrogenic exposure in sheepshead minnows, but plasma VTG clearance is concentration and time dependent and may be detected at measurable levels for months after initial exposure to an estrogenic compound.
 
Article
The effect of 17-beta estradiol (E2) and 4-nonylphenol (4-NP) on smoltification and downstream migration of Atlantic salmon was studied in an integrated laboratory and field study. In a stock of hatchery-raised 1-year-old salmon, smoltification progressed from February until late May as judged by increased gill Na+, K+ -ATPase activity and 24 h sea water (SW)-tolerance. Starting late March, three groups of 150 fish were each given 6 serial injections over 20 days of 2 microg/g body weight E2, 120 microg/g 4-NP dissolved in peanut oil or peanut oil (4 microl/g) as control. After the last injection, all fish were individually tagged (Passive Integrated Transponder tags) and a non-lethal gill biopsy was taken. Two days later (8 April), 100 fish per group were transported to the field site and released into a small stream. Smolt migration was registered by measuring arrival time at a trap downstream of the release site. Serum vitellogenin levels increased several-fold in both male and female E2- and 4-NP-treated fish. Overall, E2- and 4-NP-treatment impaired smolting as judged by elevated condition factor, reduced gill Na+, K+ -ATPase activity and alpha-subunit Na+, K+ -ATPase mRNA level, reduced muscle water content and increased mortality following 24 h SW-challenge. After release, control fish initiated downstream migration immediately, with 50% of the total number of migrants appearing in the trap within 10 days. E2- and 4-NP-treated fish appeared in the trap with a delay in comparison to controls of 6 and 8 days, respectively. After the smolt run, no fish were registered by electro-fishing upstream of the trap. The total number of fish reaching the trap and thus post-release survival was in the order control (81%), E2 (53%), 4-NP (12%). Representatives from all treatment groups held under simulated natural conditions in the laboratory survived 100% through the migration period, suggesting that a combination of behavioural and in-stream factors (predation by herons) may contribute to the differential mortality. The study indicates that short-term exposure to natural and environmental estrogens may impair smolt development and survival and delay subsequent downstream migration in Atlantic salmon.
 
Article
Results from previous experiments directed to determine the effect of different nutritional factors or the effect of xenobiotics on hormonal control of reproduction, lead to the hypothesis that hormonal perturbations repeatedly observed in sea bass (Dicentrarchus labrax) broodstock feeding commercial diets could have been caused by the presence of aryl hydrocarbon receptor (AhR) ligands, such as dioxins, furans and polychlorinated biphenyls (PCBs) in the diet. To evaluate this hypothesis, dioxins and related compounds were analysed in liver of female sea bass fed with a commercial or with a natural diet consisting of trash fish (bogue, Boops boops), and concentrations of vitellogenin (VTG) and 17beta-estradiol (E2) were determined in plasma obtained previously in monthly samplings of these animals. As observed in other experiments, females fed with a commercial diet exhibited lower VTG and higher E2 plasma levels than females fed with the natural diet. In liver, sea bass fed with the commercial diet exhibited a profile clearly dominated by high-chlorinated dioxins while in fish fed with the natural diet this profile was dominated by low chlorinated furans. However, typical AhR ligands, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin showed no differences between groups or, as is the case of planar PCBs, showed higher concentrations in the liver of fish fed with the natural diet. These results do not permit to explain the observed hormonal alterations by a possible antiestrogenic effect caused by dioxins and related compounds.
 
Article
The effect of a single injection of 17beta-estradiol (E2) was evaluated in the hermaphrodite fish Kryptolebias marmoratus. The fish [average body weight (BW), 0.15+/-0.01 g] were injected with either two concentrations of E2 (1 and 100 microg/g BW) once intraperitoneally. They were sampled at intervals of 7, 15, and 30 days after a single E2 injection. Gonadosomatic index (GSI), hepatosomatic index (HSI), the frequency of gonadal development, number of ovulated eggs, and plasma steroids levels were measured. The transcript abundances of vitellogenin (VTG) and estrogen receptors (ERalpha and beta) mRNA in the liver were also analyzed using quantitative real time polymerase chain reaction (real time PCR). GSI and the frequency of mature oocytes in the 100-microg E2-exposed group decreased compared to that of the control group during the experiment, and the number of ovulated eggs in the 100-microg E2-exposed group was lower when compared to the other groups. However, plasma E2 and 11-ketotestosterone (11-KT) levels were not significantly different between the experimental groups. On the other hand, plasma testosterone level and VTG mRNA abundance in the 100-microg E2-exposed group were significantly lower than the control group after 30 days. These results indicate that E2 stimulation at high concentration interferes with reproductive phenomena through delayed response. In addition, HSI in the 100-microg E2-exposed group and ERalpha mRNA abundance in the 1-microg E2-exposed group were significantly higher than the control group at 7 days after E2 injection, although there was no significant difference in HSI and ERalpha mRNA between all groups at 30 days. These results indicate temporal responses in reproductive parameters following high-dose E2 exposure in the hermaphrodite fish K. marmoratus.
 
Article
Genotoxicity biomarkers are widely measured in ecotoxicology as molecular toxic endpoints of major environmental pollutants. However, the long-term consequences of such damage still have to be elucidated. Some authors have suggested that the accumulation of unrepaired DNA lesions could explain the embryotoxicity of certain chemical pollutants. As embryotoxicity exerts a direct impact on the recruitment rate, genotoxicity could be closely related to disturbances of ecological concern and produce a possible impact upon population dynamics.
 
Article
The estrogenic activity of compounds was evaluated in a comparative approach both with in vitro and in vivo assays. By comparing simultaneously obtained experimental data, we evaluated the differences in response sensitivity (by EC10) and concentration-response relationships (including EC50) in order to get an idea about the predictive value of in vitro assays for in vivo estrogenic potencies or effects in fish. Two human estrogen receptor-based assays, the MVLN-assay (transformed MCF-7 human breast cancer cell line) and the yeast estrogen screen (YES-screen) were used for the in vitro evaluation of the estrogenic potencies. An in vivo model with the female zebrafish (Danio rerio) with plasma vitellogenin (VTG) as a biomarker for exposure and the ovarian somatic index (OSI) as an effect endpoint was used for the in vivo work. Compounds tested were 17beta-estradiol (E2), estrone (E1), 17alpha-ethynylestradiol (EE2) and the alkylphenolic compound nonylphenol (NP). All compounds were found to be estrogenic in both in vitro assays and were able to induce VTG and to reduce the ovarian somatic index in female zebrafish. The MVLN-assay appeared up to 15 times more sensitive than the YES-screen. Concentration-response relationships, determined by EC10 and EC50 (concentration of test compound causing 10% or 50% effect compared to control) for VTG and OSI were of the same order of magnitude, indicating that VTG induction as an exposure biomarker can be predictive for effects on ovaries in females. We further demonstrated that for E1 and NP, the in vitro observed estrogenic potencies, based on EC50 values, were of the same order of magnitude as the in vivo estrogenic potencies. For EE2, a difference between in vitro and in vivo relative estrogenic potency was observed, being about 25 times more potent in vivo than could be expected based on the in vitro results. These experimental results showed the suitability of in vitro assays for screening purposes with qualitative assessment of estrogenicity, but they meanwhile point to the need of in vivo tests for an accurate hazard assessment for wildlife.
 
Article
Considerable research has been done on the effects of endocrine disrupting chemicals (EDCs) on reproduction and gene expression in the brain, liver and gonads of teleost fish, but information on impacts to the pituitary gland are still limited despite its central role in regulating reproduction. The aim of this study was to further our understanding of the potential effects of natural and synthetic estrogens on the brain-pituitary-gonad axis in fish by determining the effects of 17α-ethynylestradiol (EE2) on the pituitary transcriptome. We exposed sub-adult coho salmon (Oncorhynchus kisutch) to 0 or 12ng EE2/L for up to 6 weeks and effects on the pituitary transcriptome of females were assessed using high-throughput Illumina(®) sequencing, RNA-Seq and pathway analysis. After 1 or 6 weeks, 218 and 670 contiguous sequences (contigs) respectively, were differentially expressed in pituitaries of EE2-exposed fish relative to control. Two of the most highly up- and down-regulated contigs were luteinizing hormone β subunit (241-fold and 395-fold at 1 and 6 weeks, respectively) and follicle-stimulating hormone β subunit (-3.4-fold at 6 weeks). Additional contigs related to gonadotropin synthesis and release were differentially expressed in EE2-exposed fish relative to controls. These included contigs involved in gonadotropin releasing hormone (GNRH) and transforming growth factor-β signaling. There was an over-representation of significantly affected contigs in 33 and 18 canonical pathways at 1 and 6 weeks, respectively, including circadian rhythm signaling, calcium signaling, peroxisome proliferator-activated receptor (PPAR) signaling, PPARα/retinoid x receptor α activation, and netrin signaling. Network analysis identified potential interactions between genes involved in circadian rhythm and GNRH signaling, suggesting possible effects of EE2 on timing of reproductive events.
 
Article
The synthetic estrogen 17-α-ethynylestradiol (EE2), a component of birth control and hormone replacement therapy, is discharged into the environment via wastewater treatment plant (WWTP) effluents. The present study employed radiolabeled EE2 to examine impacts of temperature and salinity on EE2 uptake in male killifish (Fundulus heteroclitus). Fish were exposed to a nominal concentration of 100ng/L EE2 for 2h. The rate of EE2 uptake was constant over the 2h period. Oxygen consumption rates (MO(2)), whole body uptake rates, and tissue-specific EE2 distribution were determined. In killifish acclimated to 18°C at 16ppt (50% sea water), MO(2) and EE2 uptake were both lower after 24h exposure to 10°C and 4°C, and increased after 24h exposure to 26°C. Transfer to fresh water (FW) for 24h lowered EE2 uptake rate, and long-term acclimation to fresh water reduced it by 70%. Both long-term acclimation to 100% sea water (32ppt) and a 24h transfer to 100% sea water also reduced EE2 uptake rate by 50% relative to 16ppt. Tissue-specific accumulation of EE2 was highest (40-60% of the total) in the liver plus gall bladder across all exposures, and the vast majority of this was in the bile at 2h, regardless of temperature or salinity. The carcass was the next highest accumulator (30-40%), followed by the gut (10-20%) with only small amounts in gill and spleen. Killifish chronically exposed (15 days) to 100ng/L EE2 displayed no difference in EE2 uptake rate or tissue-specific distribution. Drinking rate, measured with radiolabeled polyethylene glycol-4000, was about 25 times greater in 16ppt-acclimated killifish relative to FW-acclimated animals. However, drinking accounted for less than 30% of gut accumulation, and therefore a negligible percentage of whole body EE2 uptake rates. In general, there were strong positive relationships between EE2 uptake rates and MO(2), suggesting similar uptake pathways of these lipophilic molecules across the gills. These data will be useful in developing a predictive model of how key environmental parameter variations (salinity, temperature, dissolved oxygen) affect EE2 uptake in estuarine fish, to determine optimal timing and location of WWTP discharges.
 
Article
4-Nonylphenol (4-NP) is an endocrine disrupting substance (EDS) capable of mimicking the action of 17beta-estradiol (E2). It has been hypothesized that 4-NP in a pesticide formulation is linked to historical declines in Canadian Atlantic salmon (Salmo salar L.) populations, with effects being related to exposure during parr-smolt transformation (PST). To test this hypothesis, Atlantic salmon smolts were exposed to pulse-doses of water-borne 4-NP (20 ug/l), sustained doses of water-borne E2 (100 ng/l) (positive control), or ethanol vehicle (negative control) in mid-May during the final stages of PST. Individually tagged smolts were then sampled at three times (June, July and October) to monitor subsequent growth in sea water and plasma insulin-like growth factor I (IGF-I) concentrations. Smolt weights and plasma IGF-I concentrations were both affected by E2 and 4-NP. The effects of E2 and 4-NP on mean smolt weights were most prominent in July and October [E2 (*98.1 +/- 2.8, *242.3 +/- 10.6 g), 4-NP (*102.1 +/- 3.1, 255.7 +/- 9.5 g), controls (112.5 +/- 2.8, 282.3 +/- 8.8 g)] (P < 0.05), while their effects on mean plasma IGF-I concentrations were most prominent in June and October [E2 (15.0 +/- 1.9, 28.4 +/- 1.8 ng/ml), 4-NP (*14.8 +/- 1.9, *21.6 +/- 1.7 ng/ml), controls (20.0 +/- 1.1, 31.1 +/- 2.0 ng/ml)] (P < 0.05). Additionally, results suggest that the mechanisms of action of E2 and 4-NP involve disruption in the GH/IGF-I axis, and that they may be different from each other. The effects of E2 and 4-NP on growth and plasma IGF-I concentrations observed in this study are ecologically significant because they evoke concerns for successful growth and survival of wild salmon smolts exposed to low levels of estrogenic substances that may occur from current discharges into rivers supporting sea-run salmon stocks.
 
Article
The effects of the androgen, 17alpha-methyltestosterone were assessed on sexual development and reproductive performance in the fathead minnow (Pimephales promelas) using a gonadal recrudescence assay. In this assay, mature male and female fathead minnow, previously kept under simulated winter conditions (15 degrees C; 8:16 h light:dark regime) were transferred to simulated summer conditions (25 degrees C water temperature; 16:8 h light:dark regime) to induce gonadal recrudescence. To assess sexual development fish were exposed to nominal concentrations of 0, 0.1, 1, 5 and 50 microg/L 17alpha-methyltestosterone. After 3 weeks of chemical exposure, effects on condition (condition factor, CF), plasma vitellogenin (VTG), secondary sex characteristics, gonad growth (gonadosomatic index; GSI) and gonad histology were investigated. Reproductive performance, including reproductive output (egg production), spawning behaviour, and fertilisation rate were measured over a subsequent 3-week-period in breeding adults maintained in clean water. 17alpha-Methyltestosterone had no effects on the condition of fish at any of the doses tested. 17alpha-Methyltestosterone induced both androgenic and estrogenic effects with females generally more affected by 17alpha-methyltestosterone than males: atretic follicles and male-specific sex characteristics (androgenic effect) were induced in females at > or = 0.1 and > or = 1 microg/L 17alpha-methyltestosterone, respectively. An inhibitory effect on ovary growth occurred at an exposure concentration of 50 microg/L 17alpha-methyltestosterone. In males 1 microg/L 17alpha-methyltestosterone induced a concentration-response induction of plasma vitellogenin (estrogenic effect) likely due to its conversion into 17alpha-methylestradiol, rather to the competition with endogenous steroids and their cross reactivity with the estrogen receptor. In the fish breeding studies, concentration-dependent reductions in egg number, fertilisation rate and increases in abnormal sexual behaviour in females were observed. All of these effects occurred at exposure concentrations of > or = 5 microg/L 17alpha-methyltestosterone. Thus, it could be assumed that the observed estrogenic effects in male fathead minnow were likely to the conversion of 17alpha-methyltestosterone into the estrogen 17alpha-methylestradiol, rather to the acting of 17alpha-methyltestosterone itself. In conclusion to this, showing hormonally activity of 17alpha-methyltestosterone in fish down to 100 ng/L, indicates that its potency was close to the range of several naturally occurring estrogens.
 
Article
The synthetic estrogen, 17α-ethinylestradiol (EE2), is the active component in oral contraceptive pills. It is excreted from the human body in high amounts and released via sewage treatment plant effluent into aquatic environments. In fish, estrogen receptors have strong binding affinities for EE2, and exposure raises the possibility of adverse neuroendocrine responses in aquatic animals. In the present study we explored the effects of dissolved-phase EE2 on the dynamics of male-male aggression and courtship behaviors in adult zebrafish. Further, we assessed whether the behavioral effects of EE2 result in changes in male offspring paternity. We scored the aggressive behaviors of individual unexposed males and categorized these fish as either dominant or subordinate. We then exposed dominant males to EE2 at doses of 0, 0.5, 5.0, and 50.0 ng/L for 48 h. Subsequent trials examined the agonistic behaviors of males in two testing scenarios: (1) a dyadic encounter with another male alone, and (2) a competitive spawning interaction with another male and three adult females. Competitive spawning tests were also used to assess the impacts of EE2 exposure on courtship behavior and paternity using males that were homozygous for green fluorescent protein (GFP) expression under the control of the islet-1 promoter. We found that EE2 at all exposure concentrations reduced male aggression during male-male dyadic encounters and caused a social dominance reversal in 50% of the fish at the highest exposure dose (50 ng/L EE2). The frequency of courtship-specific behavior decreased in dominant males exposed to the steroid, though this effect was only significant for the lowest dose group (0.5 ng/L EE2). In the highest exposure group (50 ng/L EE2), 50% of dominant males relinquished paternal dominance. Our results show that short-term exposure to EE2 at environmentally relevant levels can alter aggression, and shift individual social status and reproductive success in male zebrafish.
 
Article
The sulfonation of 17beta-estradiol (E2) by human liver and recombinant sulfotransferases is influenced by environmental contaminants such as hydroxylated metabolites of polychlorinated biphenyls (OH-PCBs), which are potent inhibitors, and the therapeutic drug, celecoxib, which affects positional sulfonation of E2. In some locations, the aquatic environment is contaminated by PCBs, OH-PCBs and widely used therapeutic drugs. The objectives of this study were to investigate the sulfonation kinetics of E2 in liver cytosol from channel catfish (Ictalurus punctatus); to examine the effect of OH-PCBs on E2 sulfonation; and to determine if celecoxib altered the position of E2 sulfonation, as it does with human liver cytosol. E2 was converted to both 3- and 17-sulfates by catfish liver cytosol. At E2 concentrations below 1 microM, formation of E2-3-sulfate (E2-3-S) predominated, but substrate inhibition was observed at higher concentrations. Rates of E2-3-S formation at different E2 concentrations were fit to a substrate inhibition model, with K'm and V'max values of 0.40 +/- 0.10 microM and 91.0 +/- 4.7 pmol/min/mg protein, respectively and K(i) of 1.08 +/- 0.09 microM. The formation of E2-17-S fit Michaelis-Menten kinetics over the concentration range 25 nM to 2.5 microM, with K(m) and V(max) values of 1.07 +/- 0.23 microM and 25.7 +/- 4.43 pmol/min/mg protein, respectively. The efficiency (V(max)/K(m)) of formation of E2-3-S was 9.8-fold higher than that of E2-17-S. Several OH-PCBs inhibited E2 3-sulfonation, measured at an E2 concentration of 1 nM. Of those tested, the most potent inhibitor was 4'-OH-CB79, with two chlorine atoms flanking the OH group (IC(50): 94 nM). The inhibition of estrogen sulfonation by OH-PCBs may disrupt the endocrine system and thus contribute to the known toxic effects of these compounds. Celecoxib did not stimulate E2-17-S formation, as is the case with human liver cytosol, but did inhibit the formation of E2-3-S (IC(50): 44 microM) and to a lesser extent, E2-17-S (IC(50): > 160 microM), suggesting the previously found effect of celecoxib on E2-17-S formation may be specific to human SULT2A1.
 
Article
Juvenile medaka (Oryzias latipes) of the d-rR strain were exposed for 2 months to 1, 10 and 100 ng/l of the environmental estrogen 17-alpha-ethinylestradiol (EE(2)). The exposure period was followed by a 6 week recovering period in order to detect long-lasting effects on sexual differentiation. Survival rate, sex ratio, gonadal growth, spawning, fecundity, histology as well as the ovarian gene expression of aromatase were monitored. At 100 ng/l EE(2), all XY medaka were sex reversed and had developed an ovary. At lower EE(2) concentrations, which did not result in sex reversal, no alteration of testicular structure was detected and male fertility appeared to be unchanged. In XX females, reduced production of eggs was reflected in a significantly reduced gonadal weight at 10 and 100 ng/l EE(2). Aromatase, which in gonads is normally only expressed in ovaries, was detectable in testis of XY males exposed to 10 ng/l EE(2). The results indicated that a combination of different biological endpoints provide a suitable set of parameters for the biological evaluation of xenoestrogens, since the expression of molecular marker genes was not always paralleled by morphological deficiencies.
 
Article
Mussels (Mytilus galloaprovincialis) were exposed to different concentrations of estradiol (20, 200, and 2000 ng/L) in a semi-static regime (1-day dosing intervals) for up to 7 days in an attempt to see how mussels deal with exogenous estrogenic compounds. Whole tissue free-estradiol levels were only significantly elevated at the high exposure dose, whereas total-estradiol (free+esterified) sharply increased in a dose-dependent manner, from 2 ng/g in controls to 258 ng/g at the high exposure group. Neither free nor esterified testosterone levels showed significant differences between control and exposed organisms. The results suggest the existence of mechanisms that allow mussels to maintain their hormonal levels stable, with the exception of the high exposure dose, and the important role that fatty acid esterification, e.g. palmitoyl-CoA:estradiol acyltransferases, may play within those mechanisms. Additionally, the activity of 17beta-hydroxysteroid dehydrogenase (17beta-HSD), 5alpha-reductase, P450-aromatase, and estradiol-sulfotransferases were investigated in digestive gland microsomal and cytosolic fractions. All these activities were differently affected by estradiol exposure. Overall, the study contributes to the better knowledge of molluscan endocrinology, and defines new mechanisms of regulation of free steroid-levels in mussels.
 
Article
Exposures to ≤10ng/L of 17-α-ethinylestradiol (EE2) will reduce or shut down egg production in freshwater fish models, while mummichog (Fundulus heteroclitus), an estuarine species, are able to produce eggs at EE2 concentrations >3000ng/L. The objective of this study was to gain mechanistic insight into how mummichog are able to produce eggs during exposures to high EE2. Mummichog were exposed to 0, 50 or 250ng/L of EE2 for 14d. There were no changes in gonadosomatic index, liversomatic index, gonad development, or plasma estradiol levels after exposure to EE2. However, testosterone significantly decreased with EE2 exposures (50, 250ng/L). Microarray analysis in the liver revealed that cell processes associated with lipids were affected by EE2 at the transcriptome level. Based on the transcriptomics data, we hypothesize that mummichog are able to maintain lipid transport and uptake into the ovary and this may be associated with apolipoproteins, facilitating normal oocyte development. Novel gene regulatory networks for protein modification targets were also constructed to learn more about the potential roles of estrogens in the teleost liver. Although post-translational modifications (PTMs) are important regulatory mechanisms, the roles of PTMs in protein regulation in fish and the susceptibility of PTMs to aquatic pollutants are largely unexplored and may offer novel insight into mechanisms of endocrine disruption.
 
Article
Endocrine disrupting chemicals (EDCs) that are frequently detected in bodies of water downstream from sewage treatment facilities can have adverse impacts on fish and other aquatic organisms. To properly assess risk(s) from EDCs, tools are needed that can establish linkages from chemical exposures to adverse outcomes. Traditional methods of testing chemical exposure and toxicity using experimental animals are excessively resource- and time-consuming. In line with EPA's goal of reducing animal use in testing, these traditional screening methods may not be sustainable in the long term, given the ever increasing number of chemicals that must be tested for safety. One of the most promising ways to reduce costs and increase throughput is to use cell cultures instead of experimental animals. In accordance with National Research Council's vision on 21st century toxicity testing, we have developed a cell culture-based metabolomics approach for this application. Using a zebrafish (Danio rerio) liver cell line (ZFL), we have applied NMR-based metabolomics to investigate responses of ZFL cells exposed to 17α-ethynylestradiol (EE2). This analysis showed that metabolite changes induced by EE2 exposure agree well with known impacts of estrogens on live fish. The results of this study demonstrate the potential of cell-based metabolomics to assess chemical exposure and toxicity for regulatory application.
 
Article
A commonly used endpoint in bioassays testing the estrogenicity of chemicals is the induction of the egg yolk precursor vitellogenin (VTG) in male fish. However, relatively little is known about the kinetics of induction and elimination of VTG in fish exposed to xenoestrogens. In this study, we administered graded intra-arterial doses (0.001, 0.1, 1.0 and 10.0 mg/kg) of 17alpha-ethynylestradiol (EE(2)) to male rainbow trout via a dorsal aortic cannula which allowed repetitive blood sampling from individual fish for up to 48 days after injection. The plasma concentrations of VTG was quantified using an enzyme-linked immunosorbent assay procedure and the simultaneous concentrations of EE(2) were determined by gas chromatography-mass spectrometry. The pattern of VTG induction was similar for all doses of EE(2), with a 12-h lag-time before increase from basal levels (0.006-0.008 microg/ml), then increasing sharply to maximum levels within 7-9 days (C(max)=0.05, 711, 1521 and 2547 microg/ml VTG for the 0.001, 0.1, 1.0 and 10.0 mg/kg doses, respectively). After induction by EE(2), VTG declined mono-exponentially with an elimination half-life of 42-49 h. The half-life of VTG increased to 145 h in the 10 mg/kg treated fish. The pharmacokinetics of EE(2) were distinctly nonlinear with substantial increases in the elimination half-life with increasing dose. The plasma concentration-time profiles of EE(2) were influenced by enterohepatic recirculation that caused multiple or secondary peaks in the profiles. In a separate experiment, the pharmacokinetics of purified VTG was characterized after intra-arterial injection in trout. After direct injection of VTG, plasma levels declined tri-exponentially with an apparent steady-state volume of distribution of 837 ml/kg; total body clearance was 31.1 ml/h per kg, and the elimination half-life was 43.7 h.
 
The average infrared spectra of rainbow trout livers treated with 0 (control) (n = 8) and 22 g/L E2 (n = 6) for 3 weeks in: (A) 3600-3030 cm −1 region, (B) 3030-2800 cm −1 region (C H stretching region), and (C) 1800-800 cm −1 region (finger print region). The spectra were normalized with respect to the CH 2 asymmetric stretching band ((A) and (B)) and to the amide I band (C).
Article
Steroidal hormones produced by humans and animals are constantly being excreted into the environment. It has been demonstrated that sewage effluent discharged to surface water contains natural estrogens and synthetic estrogenic chemicals. As estrogen levels continuously increase in the aquatic environment, it is very important to have a detailed understanding of estrogens' effects on fish. In the present study, juvenile rainbow trout (Oncorhynchus mykiss) were exposed to 17beta-estradiol (E2) for 3 weeks and the effects of E2 on rainbow trout livers were investigated at the molecular level using Fourier transform infrared spectroscopy. The results revealed that E2 induced significant alterations in the liver tissues. A decrease in glycogen levels and protein concentration, and an increase in both the population of hepatic lipids, especially triglycerides, as well as the relative content of nucleic acids was observed in the E2 treated liver. In addition, a decrease in the membrane fluidity and an increase in lipid order were found in the cells of treated samples. In order to compare the effect of E2 with that of NP at molecular level, the fish were also treated with an estrogenic compound, nonylphenol (NP). The NP-treated fish liver spectra were found to be quite similar to those of E2-treated fish confirming that NP mimics the effect of E2 in immature rainbow trout.
 
Article
Waterborne 17alpha-ethinylestradiol (EE(2)) alters hormone-mediated biological indicators in fish. These alterations include increased plasma vitellogenin, increased intersex individuals, decreased egg and sperm production, reduced gamete quality, and complete feminization of male fish. Together, these observations implicate aquatic estrogens in a broad range of detrimental effects on fish reproduction and fitness. In addition to impairing reproductive processes, EE(2) is also a strong promoter of hepatic tumor formation. Since many ubiquitous, aquatic hepatocarcinogens form DNA adducts that are preferentially repaired by nucleotide excision repair (NER) processes, we hypothesized that EE(2) may exert co-carcinogenic effects by reducing an organisms ability to repair DNA adducts via this mechanism. The present study used fluorescence-based quantitative RT-PCR to examine effects of environmentally relevant concentrations of the semisynthetic estrogen, EE(2), on hepatic nucleotide excision repair (NER) gene expression. Adult male and female zebrafish (Danio rerio) were exposed to 1ng/L, 10ng/L or 100ng/L concentrations of EE(2), or to a solvent control (0.05%, v/v ethanol), for 7 days with static water renewal every 24h. Effectiveness of EE(2) exposure in the liver was confirmed by examining hepatic expression of two estrogen-responsive biomarkers, vitellogenin-1 and cytochrome P450-1A1 (CYP1A1). Quantitative analysis confirmed that exposure to 100ng/L EE(2) caused significant decreases in transcript abundance of several hepatic NER genes in male zebrafish, including XPC (>17-fold), XPA (>7-fold), XPD (>8-fold), and XPF (>8-fold). Adult female zebrafish exhibited a four-fold decreased in XPC mRNA abundance at all exposure concentrations. Decreased mRNA abundance of NER genes was also seen to a lesser degree at lower concentrations of EE(2). Adult male zebrafish showed greater reduction of hepatic NER transcript levels than their female counterparts, which is consistent with the sexually dimorphic incidence of hepatocellular carcinoma in many species. Decreased transcript levels of NER genes have been shown to be an important epidemiological marker for increased cancer risk and decreased repair capacity in humans.
 
Article
Mature male goldfish were exposed to different concentrations of the natural hormone 17beta-estradiol (E2). Two methods of exposure were employed, via ingestion at 0, 1, 10 and 100 microg/g food and via the water at 0, 1 and 10 microg/l. The fish were exposed for 24-28 days during the spawning period. The males were then paired with an artificially induced, spawning female and their sexual behaviour was observed during a 15 min period. The physiological status of the fish was also examined with respect to GSI, presence of milt and spawning tubercles and the blood plasma concentration of E2. Despite the relatively short exposure period, exposure to physiological levels of E2 was shown to severely affect the male goldfish reproductive behaviour and physiology. In conclusion, the results from this study and the ability to interpret the effects on this well-studied species, show that the effects of E2, and possibly other estrogenic EDCs, have severe effects at several vital levels of male goldfish reproduction. The results also suggests that the hormone E2 can act as an endocrine disrupting chemical (EDC) in the environment.
 
Top-cited authors
Chris Wood
  • University of British Columbia
Lars Förlin
  • University of Gothenburg
Anna Weston
  • University of Applied Sciences and Arts Northwestern Switzerland
Richard Di Giulio
  • Duke University
Jae-Seong Lee
  • Sungkyunkwan University