Anti-androgens act jointly in suppressing spiggin concentrations in androgen-primed female three-spined sticklebacks - Prediction of combined effects by concentration addition

This is the post-print version of the final paper published in Aquatic Toxicology. The published article is available from the link below. Changes resulting from the publishing process, such as peer review, editing, corrections, structural formatting, and other quality control mechanisms may not be reflected in this document. Changes may have been made to this work since it was submitted for publication. Copyright @ 2013 Elsevier B.V.Increasing attention is being directed at the role played by anti-androgenic chemicals in endocrine disruption of wildlife within the aquatic environment. The co-occurrence of multiple contaminants with anti-androgenic activity highlights a need for the predictive assessment of combined effects, but information about anti-androgen mixture effects on wildlife is lacking. This study evaluated the suitability of the androgenised female stickleback screen (AFSS), in which inhibition of androgen-induced spiggin production provides a quantitative assessment of anti-androgenic activity, for predicting the effect of a four component mixture of anti-androgens. The anti-androgenic activity of four known anti-androgens (vinclozolin, fenitrothion, flutamide, linuron) was evaluated from individual concentration-response data and used to design a mixture containing each chemical at equipotent concentrations. Across a 100-fold concentration range, a concentration addition approach was used to predict the response of fish to the mixture. Two studies were conducted independently at each of two laboratories. By using a novel method to adjust for differences between nominal and measured concentrations, good agreement was obtained between the actual outcome of the mixture exposure and the predicted outcome. This demonstrated for the first time that androgen receptor antagonists act in concert in an additive fashion in fish and that existing mixture methodology is effective in predicting the outcome, based on concentration-response data for individual chemicals. The sensitivity range of the AFSS assay lies within the range of anti-androgenicity reported in rivers across many locations internationally. The approach taken in our study lays the foundations for understanding how androgen receptor antagonists work together in fish and is essential in informing risk assessment methods for complex anti-androgenic mixtures in the aquatic environment.European Commission and Natural Environment Research Council

A catchment-based study of endocrine disruption in surface waters: multivariate evaluation of the health of a sentinel fish species exposed to sewage treatment works effluent

Summary of the results in the context of EDCAT 5 project aims: 1. By comparing appropriate biomarkers in fish sampled from STW-impacted sites and control sites during the pre-remediation period, to determine whether there was evidence for any effects that might be attributed to the presence of estrogenic (or androgenic, or anti-androgenic/-estrogenic) endocrine disrupting chemicals in the former. This aim was addressed by measuring concentrations of the estrogen-dependent yolk protein precursor vitellogenin, and the androgen-dependent nest glue spiggin in male and female sticklebacks. In addition histological examination of the gonadal structure of fish captured at the impacted and non-impacted sites was employed to seek evidence of overt alterations in reproductive physiology of the fish. For a subset of matched samples from the two rivers, the relative induction of hepatic choriogenin mRNA, a biomarker of estrogen exposure, was measured. Conclusions: Chemistry data provided by EDCAT3&4 showed that estrogenicity of the effluent was low prior to remediation and lower still following installation of the GAC plant. No evidence of overt estrogenic effects was detected in male sticklebacks in the Ray, VTG and ChG levels were similar in males from both rivers. Nor was there any evidence of alterations in spiggin concentrations in the kidneys of males from the Ray compared to the Ock. However, VTG concentrations in female sticklebacks from the Ray were increased following the STW upgrade as were hepatic ChG transcript levels, and kidney spiggin concentrations. No changes in these elements of the reproductive system were observed in females from the Ock across the same time periods. Chemical analysis of the effluent indicated that prior to installation of the GAC plant substantial concentrations of anti-androgenic chemicals were present, together with a wide range of other organics. Concentrations of these were much reduced following the plant upgrade. It is reasonable to suppose that the changes observed in the female reproductive endocrine system following the upgrade were related to the removal of some or all of this complex mixture of chemicals. The absence of effects in males may be related to the balance between exogenous and endogenous signals, or to the specificity of effects exerted by the chemicals present. No intersex fish were detected from either river. A significant bias in favour of females was detected in the stickleback populations in both rivers suggesting a factor associated with life-history of the fish, rather than contaminant burden, was responsible. 2. By comparing appropriate biomarkers in fish sampled from STW-impacted sites and control sites during the pre-remediation period, to determine whether there was evidence for any effects that might be attributed to the presence of “conventionally” toxic chemicals. This was addressed by measurement of the activity of a key Phase I transforming enzyme in the liver of fish, either using direct enzymatic assay (EROD) or by quantifying the levels of expression of the corresponding gene (CYP1A). Conclusions: EROD activity was significantly greater in fish from the Ray than the Ock in two samples collected prior to the installation of the GAC plant (2006, 2007) and this likely reflects the differential contaminant loading in the two rivers. A single sample following the commissioning of the GAC plant (2008) indicated that EROD activity had increased among fish from the Ock while that in fish from the Ray remained unchanged. While a delayed recovery of this biomarker in fish from the Ray may be expected depending on the route of exposure (direct via water or indirect via contaminated food) the reasons for elevated EROD activity in fish from the Ock/Childrey Brook are not immediately evident. Provision of a full data set for Cyp1A expression awaits the repeat of the assay. When this is complete the factors underlying the EROD findings may become clear. 3. To determine whether the adaptive capacity and energetic status of fish varied between the STW-impacted and non-impacted sites. This was addressed by measurement of indicators of stress (whole-body corticosteroid levels), metabolic status (whole-body glucose levels) and anabolic activity (RNA:DNA ratios). Conclusions: The data provide no evidence that the stress response of fish captured in the Ray prior to installation of the GAC plant was modified by exposure to the effluent. However, large variations in whole-body corticosteroid and glucose concentrations in fish from both rivers, with clear trends over time, were closely linked to perturbations in the river flow regime. Whether there was interaction between environmental and chemical factors in determining corticosteroid and glucose status is difficult to discern but it seems likely that variation in these indicators of the stress axis was driven primarily by environmental factors. The RNA:DNA ratios were closely linked with seasonal change in temperature and closely matched observed patterns of weight and length gain in stickleback populations in the two rivers. The longer growth period enjoyed by fish in the Ray was clearly evident. For both rivers, mean anabolic activity was greater during 2008 than 2007 and it seems likely that this is related to adverse effects associated with the periods of extreme flow change observed on both rivers in 2007. 4. To assess whether there were differences in population size and structure between STW-impacted and non-impacted sites. This was addressed by comparison of key somatic measures, in particular frequency distributions for fork length. Conclusions: Because of the extreme patchiness of the distribution of stickleback populations in both rivers accurate abundance estimates were not obtained. However, the catch per unit effort across the life of the project was similar for both rivers. While population size, and age structure (both rivers hosted annual populations), appeared to be similar fish in the Ray were overall larger than those from the Ock, and spawned earlier. The differences in growth and timing of spawning between the rivers were likely to have been associated with the Rodbourne STW effluent. Downstream of the discharge on the Ray water temperatures were consistently 2 – 3oC above those of the Ock. This temperature difference, in combination with the introduction of additional nutrients into the river which is likely to have affected the availaibility of food, probably accounts for the different growth profile among the sticklebacks in the two rivers. However, over and above this difference, there was a significant increase in size of sticklebacks in the Ray between the matched pre- and post-remediation periods in the Ray while no change in size of the fish in the Ock occurred during the same period. Similarly, the RNA:DNA ratio was higher in fish from the Ock during 2007 but greater in fish from the Ray during 2008. Taken together, these observations suggest that there was an improvement in the status of the fish in the Ray following the commissioning of the GAC plant, while the population in the Ock remained relatively stable. It is reasonable to suppose that this may be linked to the reduction of the chemical load entering the Ray at Rodbourne following the installation of the GAC plant. The Ray is “cleaner” now than was the case prior to remediation but remains nutrient rich and several degrees warmer than the Ock, this combination of factors providing fish in the Ray with greater scope for growth relative to populations in the Ock

Evidence suggesting that di-n-butyl phthalate has anti-androgenic effects in fish

This article is the pre-print version of the full and final published article.Phthalate ester plasticizers are anti-androgenic in mammals. High doses of certain phthalates consistently interfere with the normal development of male offspring exposed in utero, causing disrupted sperm production, abnormal development of the genitalia, and in some cases infertility. In the environment, phthalates are considered ubiquitous and are commonly measured in aquatic ecosystems at low ng to mu g per litre concentrations. Given the similarity between mammalian and teleost endocrine systems, phthalate esters may be able to cause anti-androgenic endocrine disruption in fish in the wild. In the present study, adult male three-spined sticklebacks (Gasterosteus aculetaus) (n = 8) were exposed to di-n-butyl phthalate (DBP) (0, 15, and 35 mu g DBP/L) for 22 d and analyzed for changes in nesting behavior, plasma androgen concentrations, spiggin concentrations, and steroidogenic gene expression. Plasma testosterone concentrations were significantly higher in males from the 35 mu g DBP/L group compared with the solvent control, whereas plasma 11-ketotestosterone concentrations were not significantly affected. Expression of steroid acute regulatory protein and 3 beta-hydroxysteroid dehydrogenase remained unchanged. Spiggin concentrations were significantly lower in the males exposed to 35 mu g DBP/L. Nest building appeared to be slower in some males exposed to DBP, but this was not statistically significant. These results suggest that DBP has anti-androgenic effects in fish. However, further research is required to firmly establish the consequences of chronic DBP exposure in fish

Relationship between Sex Steroid and Vitellogenin Concentrations in Flounder (Platichthys flesus) Sampled from an Estuary Contaminated with Estrogenic Endocrine-Disrupting Compounds

High concentrations of vitellogenin (VTG; egg yolk protein) have previously been found in male flounder (Platichthys flesus) from several UK estuaries; these levels have been ascribed to the presence of estrogenic endocrine-disrupting compounds (EDCs). Gonadal abnormalities, including intersex, have also been recorded in these estuaries. However, there is no firm evidence to date that these two findings are causally linked or that the presence of estrogenic EDCs has any adverse population effects. In the present study, we examined the relationship between concentrations of VTG and sex steroids (11-oxotestosterone in males and 17β-estradiol in females) in specimens of flounder captured from the estuary of the River Mersey. We first questioned whether the high concentrations of VTG in male and immature female flounder were indeed caused by a direct effect of exogenous EDCs and not indirectly via the endogenous secretion of 17β-estradiol. The data favored the direct involvement of estrogenic EDCs. We then questioned whether the presence of estrogenic EDCs not only stimulated inappropriate VTG synthesis but whether it might also have had a negative effect on endogenous steroid secretion. It should be noted that the predicted consequences of a drop in steroid secretion include smaller gonads, smaller oocytes, fewer numbers of sperm, and depressed spawning behavior. This question was more difficult to answer because of the strong effect of the seasonal reproductive cycle and stage of maturation on steroid concentrations. However, matched by month of capture and stage of maturation, both 17β-estradiol in females and 11-keto-testosterone in males were in most cases significantly lower in those years when VTG concentrations were higher

Sublethal exposure to copper supresses the ability to acclimate to hypoxia in a model fish species

This is the final version. Available on open access from Elsevier via the DOI in this recordHypoxia is one of the major threats to biodiversity in aquatic systems. The association of hypoxia with nutrient-rich effluent input into aquatic systems results in scenarios where hypoxic waters could be contaminated with a wide range of chemicals, including metals. Despite this, little is known about the ability of fish to respond to hypoxia when exposures occur in the presence of environmental toxicants. We address this knowledge gap by investigating the effects of exposures to different levels of oxygen in the presence or absence of copper using the three-spined sticklebacks (Gasterosteus aculeatus) model. Fish were exposed to different air saturations (AS; 100%, 75% and 50%) in combination with copper (20 μg/L) over a 4 day period. The critical oxygen level (Pcrit), an indicator of acute hypoxia tolerance, was 54.64 ± 2.51% AS under control conditions, and 36.21 ± 2.14% when fish were chronically exposed to hypoxia (50% AS) for 4 days, revealing the ability of fish to acclimate to low oxygen conditions. Importantly, the additional exposure to copper (20 μg/L) prevented this improvement in Pcrit, impairing hypoxia acclimation. In addition, an increase in ventilation rate was observed for combined copper and hypoxia exposure, compared to the single stressors or the controls. Interestingly, in the groups exposed to copper, a large increase in variation in the measured Pcrit was observed between individuals, both under normoxic and hypoxic conditions. This variation, if observed in wild populations, may lead to selection for a tolerant phenotype and alterations in the gene pool of the populations, with consequences for their sustainability. Our findings provide strong evidence that copper reduces the capacity of fish to respond to hypoxia by preventing acclimation and will inform predictions of the consequences of global increases of hypoxia in water systems affected by other pollutants worldwide.University of ExeterCentre for Environment Fisheries and Aquaculture Science (Cefas)Biotechnology and Biological Sciences Research Council (BBSRC)ONICYT-FONDECY

Baseline data from the EDCAT research programme on oestrogens and fish populations

The EDCAT programme now has over two year’s baseline data on the chemistry and fish biology of an English river, the Ray, which receives oestrogenic sewage effluent from the city of Swindon. It has developed models which predict oestrogen concentrations in the river throughout the year, and has successfully corroborated these with data on oestrogenic substances, in vitro oestrogenic activity, and non-oestrogenic contaminants. It has also gathered data on the dynamics and oestrogenic responses of stickleback populations in the river during a period when the sewage discharge received both normal dilution, and high dilution from unusually large river flows. Finally, breeding experiments with wild intersex roach have been conducted in which the breeding success of each individual has been tracked by microsatellite analysis of offspring. The Swindon (Rodbourne) sewage treatment plant has now been upgraded with granular activated carbon filtration technology to remove oestrogens, and future work of the EDCAT programme will monitor the rate of recovery of fish populations in the River Ray

Hypoxia modifies the response to flutamide and linuron in male three-spined stickleback (Gasterosteus aculeatus)

This is the author accepted manuscript. The final version is available from Elsevier via the DOI in this recordHypoxia is a major stressor in aquatic environments and it is frequently linked with excess nutrients resulting from sewage effluent discharges and agricultural runoff, which often also contain complex mixtures of chemicals. Despite this, interactions between hypoxia and chemical toxicity are poorly understood. We exposed male three-spined stickleback during the onset of sexual maturation to a model anti-androgen (flutamide; 250μg/L) and a pesticide with anti-androgenic activity (linuron; 250μg/L), under either 97% or 56% air saturation (AS). We assessed the effects of each chemical, alone and in combination with reduced oxygen concentration, by measuring the transcription of spiggin in the kidney, as a marker of androgen signalling, and 11 genes in the liver involved in some of the molecular pathways hypothesised to be affected by the exposures. Spiggin transcription was strongly inhibited by flutamide under both AS conditions. In contrast, for linuron, a strong inhibition of spiggin was observed under 97% AS, but this effect was supressed under reduced air saturation, likely due to interactions between the hypoxia inducible factor and the aryl hydrocarbon receptor (AhR) pathways. In the liver, hypoxia inducible factor 1α was induced following exposure to both flutamide and linuron, however this was independent of the level of air saturation. This work illustrates the potential for interactions between hypoxia and pollutants with endocrine or AhR agonist activity to occur, with implications for risk assessment and management.Centre for Environment, Fisheries and Aquaculture Scienc

Use of the Three-Spined Stickleback (Gasterosteus aculeatus) As a Sensitive in Vivo Test for Detection of Environmental Antiandrogens

We have previously shown that exposure to exogenous androgens causes female sticklebacks (Gasterosteus aculeatus) to produce the glue protein, spiggin, in their kidneys. This protein can be quantified by an enzyme-linked immunosorbent assay developed and validated at the Centre for Environment, Fisheries and Aquaculture Science. Here we report the development of an in vivo test for the detection of environmental antiandrogens. The system involves the simultaneous exposure of female sticklebacks to 17α-methyltestosterone (a model androgen) at 500 ng/L and suspected environmental antiandrogens over a period of 21 days. The spiggin content of the kidneys is then measured, and any antiandrogenic activity is evaluated by comparing the spiggin levels of female fish exposed to antiandrogens to those of female fish exposed solely to the model androgen. The assay detects the antiandrogenic activity of flutamide, vinclozolin (both used at 250 μg/L), linuron (at 150 μg/L), and fenitrothion (at 15 and 150 μg/L). These results provide the first evidence of in vivo antiandrogenic activity of both linuron and fenitrothion in teleosts. Although there are other suggested fish species that could be used for this purpose, the stickleback is the only widely available species in which it is now possible to study both estrogenic and antiandrogenic end points in the same individual. Furthermore, the species is endemic and ubiquitous in Europe, and it possesses many ecological traits that make it better suited than other potential species for field research into endocrine disruption

Hepatic transcriptional responses to copper in the three-spined stickleback are affected by their pollution exposure history

This is the author accepted manuscript. The final version is available from the publisher via the DOI in this record.Some fish populations inhabiting contaminated environments show evidence of increased chemical tolerance, however the mechanisms contributing to this tolerance, and whether this is heritable, are poorly understood. We investigated the responses of two populations of wild three-spined stickleback (Gasterosteus aculeatus) with different histories of contaminant exposure to an oestrogen and copper, two widespread aquatic pollutants. Male stickleback originating from two sites, the River Aire, with a history of complex pollution discharges, and Siblyback Lake, with a history of metal contamination, were depurated and then exposed to copper (46μg/L) and the synthetic oestrogen ethinyloestradiol (22ng/L). The hepatic transcriptomic response was compared between the two populations and to a reference population with no known history of exposure (Houghton Springs, Dorset). Gene responses included those typical for both copper and oestrogen, with no discernable difference in response to oestrogen between populations. There was, however, some difference in the magnitude of response to copper between populations. Siblyback fish showed an elevated baseline transcription of genes encoding metallothioneins and a lower level of metallothionein induction following copper exposure, compared to those from the River Aire. Similarly, a further experiment with an F1 generation of Siblyback fish bred in the laboratory found evidence for elevated transcription of genes encoding metallothioneins in unexposed fish, together with an altered transcriptional response to 125μg/L copper, compared with F1 fish originating from the clean reference population exposed to the same copper concentration. These data suggest that the stickleback from Siblyback Lake have a differential response to copper, which is inherited by the F1 generation in laboratory conditions, and for which the underlying mechanism may include an elevation of baseline transcription of genes encoding metallothioneins. The genetic and/or epigenetic mechanisms contributing to this inherited alteration of metallothionein transcription have yet to be established.This work was funded by the UK NERC postgenomic and proteomic programme grant NE/C507661/1 and by a Fisheries Society of the British Isles research grant to EMS. Birmingham functional genomics facilities were funded by BBSRC grant 6/JIF13209. We thank R.E. Godfrey, S. Jondhale, A. Jones, and L. Klovrza for technical assistance, J.K. Chipman for help and support, and the Environment Agency for provision of water chemistry data