Anke Lange

Alterations of tissue glutathione levels and metallothionein mRNA in rainbow trout during single and combined exposure to cadmium and zinc.

The objective of this study was to assess the effects of Cd and Zn exposure of rainbow trout (Oncorhynchus mykiss) on (a) hepatic glutathione (GSH) levels; and (b) hepatic and branchial metallothionein (MT) mRNA expression. Juvenile rainbow trout were exposed to waterborne Cd (nominal concentrations: 1.5 or 10 microg Cd l(-1)), Zn (150 or 1000 microg Zn l(-1)) or Cd/Zn mixtures (1.5 microg Cd l(-1) with 200 microg Zn l(-1) or 10 microg Cd l(-1) with 1000 microg Zn l(-1)). After 14 and 28 days of treatment, hepatic concentrations of total glutathione, oxidized glutathione (GSSG) and cysteine were determined by means of fluorometric high performance liquid chromatography (HPLC). Branchial and hepatic expression of MT mRNA was measured by means of semi-quantitative RT-PCR. Exposure of trout to Zn did not result in significantly elevated tissue levels of Zn, whereas Cd accumulation factors changed significantly with time and concentration. Despite of the absence of Zn accumulation, hepatic GSH but not MT mRNA levels were significantly altered in Zn-exposed fish. Cd, on the contrary, affected mainly the MT response but not GSH. Also tissue specific differences in the regulation of the two thiol pools were expressed. The thiol response after exposure to metal mixtures could not be explained by simple addition of the effects of the individual metals. The results indicate that cellular thiol pools show different reaction patterns with respect to specific metals and metal mixtures. Under conditions of long-term, low dose metal exposure, the function of GSH appears to go beyond that of a transitory, first line defense.

Molecular Mechanisms of Toxicity of Silver Nanoparticles in Zebrafish Embryos

Silver nanoparticles cause toxicity in exposed organisms and are an environmental health concern. The mechanisms of silver nanoparticle toxicity, however, remain unclear. We examined the effects of exposure to silver in nano-, bulk-, and ionic forms on zebrafish embryos (Danio rerio) using a Next Generation Sequencing approach in an Illumina platform (High-Throughput SuperSAGE). Significant alterations in gene expression were found for all treatments and many of the gene pathways affected, most notably those associated with oxidative phosphorylation and protein synthesis, overlapped strongly between the three treatments indicating similar mechanisms of toxicity for the three forms of silver studied. Changes in oxidative phosphorylation indicated a down-regulation of this pathway at 24 h of exposure, but with a recovery at 48 h. This finding was consistent with a dose-dependent decrease in oxygen consumption at 24 h, but not at 48 h, following exposure to silver ions. Overall, our data provide support for the hypothesis that the toxicity caused by silver nanoparticles is principally associated with bioavailable silver ions in exposed zebrafish embryos. These findings are important in the evaluation of the risk that silver particles may pose to exposed vertebrate organisms.

Evidence for the existence of a functional Kiss1/Kiss1 receptor pathway in fish

In mammals, the Kiss1 receptor (Kiss1r) and its kisspeptin ligands are key factors regulating the onset of puberty. In fish, however, the mechanisms underlying the initiation of puberty are poorly understood and the role of the Kiss1r/kisspeptin pathway in this process has not been established. In this study, a bioinformatics approach was used to identify the genes for Kiss1 and Kiss1r in five teleost genomes and the information used to clone the corresponding transcripts from zebrafish. Zebrafish kiss1r was expressed predominantly in the brain, with a minor level of expression in the eye, and zebrafish kiss1 was expressed in brain, intestine, adipose tissue and testis. Analysis of the chromosome region containing the kiss1 locus showed high synteny across vertebrate genomes. In contrast to their mammalian homologues, teleost Kiss1 protein sequences were poorly conserved with the exception of the region representing kisspeptin-10. Signal peptide sequences and likely cleavage and amidation sites in the teleost Kiss1 sequences were determined and found to be similar to those in mammalian Kiss1. This is the first report of the existence and characterization of the Kiss1 gene outside the mammalian taxa, suggesting that a functional Kiss1/Kiss1 receptor pathway is conserved across vertebrate species.

Bioassay-directed identification of novel antiandrogenic compounds in bile of fish exposed to wastewater effluents

The widespread occurrence of feminized male fish downstream of some UK Wastewater Treatment Works (WwTWs) has been associated with exposure to estrogenic and potentially antiandrogenic (AA) contaminants in the effluents. In this study, profiling of AA contaminants in WwTW effluents and fish was conducted using HPLC in combination with in vitro androgen receptor transcription screens. Analysis of extracts of wastewater effluents revealed complex profiles of AA activity comprising 21-53 HPLC fractions. Structures of bioavailable antiandrogens were identified by exposing rainbow trout to a WwTW effluent and profiling the bile for AA activity using yeast (anti-YAS) and mammalian-based (AR-CALUX) androgen receptor transcription screens. The predominant fractions with AA activity in both androgen receptor screens contained the germicides chlorophene and triclosan, and together these contaminants accounted for 51% of the total anti-YAS activity in the fish bile. Other AA compounds identified in bile included chloroxylenol, dichlorophene, resin acids, napthols, oxybenzone, 4-nonylphenol, and bisphenol A. Pure standards of these compounds were active in the androgen receptor screens at potencies relative to flutamide of between 0.1 and 13.0. Thus, we have identified, for the first time, a diverse range of AA chemicals in WwTWs that are bioavailable to fish and which need to be assessed for their risk to the reproductive health of these organisms and other aquatic biota.

Implications of Persistent Exposure to Treated Wastewater Effluent for Breeding in Wild Roach (Rutilus rutilus) Populations

Feminized responses are widespread in wild populations of roach, Rutilus rutilus, living in UK rivers, and some of these responses have been shown to arise as a consequence of exposure to wastewater treatment works (WwTW) effluent discharges and the endocrine disrupting chemicals (EDCs) they contain. The causation of the ovotestis condition in wild roach, however, has yet to be established. Furthermore, the impact of long-term exposure to WwTW effluents on the reproductive fitness of wild fish populations is not known, and this information is crucial for population level effect assessments. We undertook a chronic exposure of roach to a treated estrogenic wastewater effluent for up to 3.5 years to assess principally for effects on subsequent reproductive fitness, as determined through parentage analysis on offspring from a competitive breeding study. In generating the fish for the breeding study we found that exposure to full strength WwTW effluent until sexual maturity resulted in sex reversal in almost all males in the population; 98% of the exposed fish were phenotypic females, containing ovaries. Furthermore, fish exposed to a 50% dilution of WwTW effluent contained ovotestis (21% of the male roach) that was absent from the control population. In competitive breeding studies, and applying DNA microsatellites to assess parentage, we show that presumptive females exposed to sexual maturity to WwTW effluent bred normally, albeit in the absence of nonexposed females, but putative sex-reversed males breeding as females contributed poorly, if at all, in a breeding population, depending on the competition. These novel findings on sex reversal add a new dimension for impact assessments of exposure to WwTW effluents on fish populations.

Altered Sexual Development in Roach (Rutilus rutilus) Exposed to Environmental Concentrations of the Pharmaceutical 17α-Ethinylestradiol and Associated Expression Dynamics of Aromatases and Estrogen Receptors

Wild roach (Rutilus rutilus) inhabiting UK rivers contaminated with estrogenic effluents from wastewater treatment works show altered sexual development, including intersex, and this can impact negatively on their reproductive capabilities. The molecular events underlying these disruptions in gender assignment, however, are still poorly understood. In this study, two isoforms of aromatase (cyp19a1a and cyp19a1b) were cloned from the roach, and effects of exposure to 17alpha-ethinylestradiol (EE(2)) during early life were determined on the expression of both aromatases and on the estrogen receptors (ERs) (subtypes esr1 and esr2b) and analyzed against effects on the progression of gonadal sex differentiation. Exposure to environmentally relevant concentrations of EE(2) during the critical period of sex differentiation resulted in gonadal feminization and all roach exposed to 4 ng EE(2)/l were females. These effects on gonadal development were associated with alterations in the expression of both esr and cyp19a1 genes in bodies and heads of exposed fish with the most marked effects on the expression of esr1 and cyp19a1b. Our findings show that both aromatase isoforms and both ER subtypes are associated with sexual differentiation in roach, and alterations in their expression can signal for disruptions in sexual development.

Comparative responsiveness to natural and synthetic estrogens of fish species commonly used in the laboratory and field monitoring

Exposure to estrogenic chemicals discharged into the aquatic environment has been shown to induce feminization in wild freshwater fish and although fish species have been reported to differ in their susceptibility for these effects, empirical studies that directly address this hypothesis are lacking. In this study, in vitro ERα activation assays were applied in a range of fish species used widely in chemical testing (including, zebrafish, fathead minnow, medaka) and/or as environmental monitoring species (including, roach, stickleback, carp) to assess their comparative responsiveness to natural (estrone, estradiol, estriol) and synthetic (17α-ethinylestradiol (EE2), diethylstilbestrol (DES)) estrogens. In vivo exposures to EE2 via the water (nominal 2 and 10 ng/L for 7 days) were also conducted for seven fish species to compare their responsiveness for hepatic vitellogenin (VTG) mRNA induction (an ER mediated response). Of the fish species tested, zebrafish ERα was found to be the most responsive and carp and stickleback ERα the least responsive to natural steroid estrogens. This was also the case for exposure to EE2 with an ERα-mediated response sensitivity order of zebrafish > medaka > roach > fathead minnow > carp > stickleback. For VTG mRNA induction in vivo, the order of species responsiveness was: rainbow trout (not tested in the ERα activation assays) > zebrafish > fathead minnow > medaka > roach > stickleback > carp. Overall, the responses to steroid estrogens in vitro via ERα compared well with those seen in vivo (VTG induction for exposure to EE2) showing in vitro screening of chemicals using fish ERα-mediated responses indicative of estrogenic responses (VTG induction) in vivo.

Differing species responsiveness of estrogenic contaminants in fish is conferred by the ligand binding domain of the estrogen receptor.

Exposure to estrogenic endocrine disrupting chemicals (EDCs) induces a range of adverse effects, notably on reproduction and reproductive development. These responses are mediated via estrogen receptors (ERs). Different species of fish may show differences in their responsiveness to environmental estrogens but there is very limited understanding on the underlying mechanisms accounting for these differences. We used custom developed in vitro ERα reporter gene assays for nine fish species to analyze the ligand- and species-specificity for 12 environmental estrogens. Transcriptonal activities mediated by estradiol-17β (E2) were similar to only a 3-fold difference in ERα sensitivity between species. Diethylstilbestrol was the most potent estrogen (∼ 10-fold that of E2) in transactivating the fish ERαs, whereas equilin was about 1 order of magnitude less potent in all species compared to E2. Responses of the different fish ERαs to weaker environmental estrogens varied, and for some considerably. Medaka, stickleback, bluegill and guppy showed higher sensitivities to nonylphenol, octylphenol, bisphenol A and the DDT-metabolites compared with cyprinid ERαs. Triclosan had little or no transactivation of the fish ERαs. By constructing ERα chimeras in which the AF-containing domains were swapped between various fish species with contrasting responsiveness and subsequent exposure to different environmental estrogens. Our in vitro data indicate that the LBD plays a significant role in accounting for ligand sensitivity of ERα in different species. The differences seen in responsiveness to different estrogenic chemicals between species indicate environmental risk assessment for estrogens cannot necessarily be predicted for all fish by simply examining receptor activation for a few model fish species.

Metabolomics reveals target and off-target toxicities of a model organophosphate pesticide to roach (Rutilus rutilus): implications for biomonitoring.

The ability of targeted and nontargeted metabolomics to discover chronic ecotoxicological effects is largely unexplored. Fenitrothion, an organophosphate pesticide, is categorized as a “red list” pollutant, being particularly hazardous to aquatic life. It acts primarily as a cholinesterase inhibitor, but evidence suggests it can also act as an androgen receptor antagonist. Whole-organism fenitrothion-induced toxicity is well-established, but information regarding target and off-target molecular toxicities is limited. Here we study the molecular responses of male roach (Rutilus rutilus) exposed to fenitrothion, including environmentally realistic concentrations, for 28 days. Acetylcholine was assessed in brain; steroid metabolism was measured in testes and plasma; and NMR and mass spectrometry-based metabolomics were conducted on testes and liver to discover off-target toxicity. O-demethylation was confirmed as a major route of pesticide degradation. Fenitrothion significantly depleted acetylcholine, confirming its primary mode of action, and 11-ketotestosterone in plasma and cortisone in testes, showing disruption of steroid metabolism. Metabolomics revealed significant perturbations to the hepatic phosphagen system and previously undocumented effects on phenylalanine metabolism in liver and testes. On the basis of several unexpected molecular responses that were opposite to the anticipated acute toxicity, we propose that chronic pesticide exposure induces an adapting phenotype in roach, which may have considerable implications for interpreting molecular biomarker responses in field-sampled fish.

Effects of pharmaceuticals on the expression of genes involved in detoxification in a carp primary hepatocyte model.

Fish in many surface freshwaters are exposed to a range of pharmaceuticals via wastewater treatment works effluent discharges. In mammals the pregnane X receptor (PXR) plays a key role in the regulation of a suite of genes involved in drug biotransformation, but information on the role of this response pathway in fish is limited. Here we investigated the effects of exposure of carp (Cyprinus carpio) primary hepatocytes to the human PXR agonist rifampicin (RIF) on expression of target genes involved in phase I (cyp2k, cyp3a) and phase II (gstα, gstπ) drug metabolism and drug transporters mdr1 and mrp2. RIF induced expression of all target genes measured and the PXR antagonist ketoconazole (KET) inhibited responses of cyp2k and cyp3a. Exposure of the primary carp hepatocytes to the pharmaceuticals ibuprofen (IBU), clotrimazole (CTZ), clofibric acid (CFA) and propranolol (PRP), found responses to IBU and CFA, but not CTZ or PRP. This is in contrast with mammals, where CTZ is a potent PXR-agonist. Collectively our data indicate potential PXR involvement in regulating selected genes involved in drug metabolism in fish, but suggest some divergence in the regulation pathways with those in mammals. The carp primary hepatocyte model serves as a useful system for screening for responses in these target genes involved in drug metabolism.