Ioanna Katsiadaki

Integrating omic technologies into aquatic ecological risk assessment and environmental monitoring: hurdles, achievements, and future outlook.

Background In this commentary we present the findings from an international consortium on fish toxicogenomics sponsored by the U.K. Natural Environment Research Council (Fish Toxicogenomics—Moving into Regulation and Monitoring, held 21–23 April 2008 at the Pacific Environmental Science Centre, Vancouver, BC, Canada). Objectives The consortium from government agencies, academia, and industry addressed three topics: progress in ecotoxicogenomics, regulatory perspectives on roadblocks for practical implementation of toxicogenomics into risk assessment, and dealing with variability in data sets. Discussion Participants noted that examples of successful application of omic technologies have been identified, but critical studies are needed to relate molecular changes to ecological adverse outcome. Participants made recommendations for the management of technical and biological variation. They also stressed the need for enhanced interdisciplinary training and communication as well as considerable investment into the generation and curation of appropriate reference omic data. Conclusions The participants concluded that, although there are hurdles to pass on the road to regulatory acceptance, omics technologies are already useful for elucidating modes of action of toxicants and can contribute to the risk assessment process as part of a weight-of-evidence approach.

Identifying health impacts of exposure to copper using transcriptomics and metabolomics in a fish model.

Copper (Cu) is a micronutrient essential for the biochemical functioning of numerous processes in vertebrates but is also often present in the aquatic environment at concentrations able to cause adverse health effects in aquatic organisms. This study investigated the signaling pathways mediating the effects of exposure to Cu using a toxicogenomic approach in a fish model, the stickleback ( Gasterosteus aculeatus ). Freshwater-acclimated male fish were exposed via the water to Cu, including at environmentally relevant concentrations (3.2-128 microg of Cu/L for 4 days), and the biological responses explored through analyses of the hepatic transcriptome and metabolome and phenotypic end points, including assessment of DNA damage in blood cells. The Cu exposures resulted in DNA strand breaks in blood cells at all exposure concentrations and alterations in hepatic gene expression and metabolite concentrations in a concentration-dependent manner (from 10 microg of Cu/L). Genes associated with the cholesterol biosynthesis pathway were significantly over-represented and consistently down-regulated (at 128 microg of Cu/L), similar to that occurring in a mouse model for Wilsons disease. Additionally, inductions in metallothionein and catalase were also observed. The concentrations of NAD(+) and lactate increased significantly with the Cu exposure, consistent with a shift toward anaerobic metabolism, and these aligned closely with changes observed in gene expression. The pathways of Cu toxicity identified in our study support the conserved mechanisms of Cu toxicity from lower vertebrates to mammals, provide novel insights into the deleterious effects of Cu in fish, and further demonstrate the utility of fish as environmental sentinels for chemical impacts on both environmental and human health.

SURVEYS OF PLASMA VITELLOGENIN AND INTERSEX IN MALE FLOUNDER (PLATICHTHYS FLESUS ) AS MEASURES OF ENDOCRINE DISRUPTION BY ESTROGENIC CONTAMINATION IN UNITED KINGDOM ESTUARIES: TEMPORAL TRENDS, 1996 TO 2001

Plasma vitellogenin (VTG) concentrations and the presence of the ovo-testis (intersex) condition have been recorded in male flounder (Platichthys flesus) captured from several United Kingdom (UK) estuaries since 1996 as part of the endocrine disruption in the Marine Environment (EDMAR) project and earlier programs. It has been confirmed that plasma VTG concentrations in male flounder have remained elevated in several UK estuaries (e.g., Tees, Mersey, and Tyne) throughout the period covered by this study. However, the time-series data indicate that plasma VTG, a measure of environmental estrogen contamination, has decreased in fish captured from several estuaries, especially those of the Tyne and Mersey. Shorter time-series data sets from the Forth and Clyde estuaries also suggest a decrease in estrogen contamination at these sites. Trends associated with specific point sources of estrogenic contamination show site-specific patterns. For instance, plasma VTG levels in male flounder captured near the Howdon sewage treatment outfall (Tyne) have shown a steady decline to near baseline levels in 2001, while the plasma of male fish captured at a site adjacent to the Dabholm Gut discharge in the Tees estuary have shown little evidence of a sustained decline. The occurrence of the intersex condition was detected at a low but consistent prevalence through the study period, with the majority of cases recorded in fish captured from the Tyne and Mersey estuaries. The data set does not allow conclusions to be drawn about any temporal trends associated with this condition. The significance of the findings and possible mitigating influences are discussed in terms of the impacts on wild fish and the role of effluent treatment in reducing these.

The impact of oestrogenic and androgenic contamination on marine organisms in the United Kingdom: summary of the EDMAR programme

This paper summarises results of the EDMAR programme which is investigating oestrogenic and androgenic endocrine disruption in UK coastal waters. Most of the data concern fish. Four species (flounder, viviparous blenny and two sand gobies) are experiencing feminisation in industrialised estuaries. In males this includes vitellogenin (VTG) synthesis, ovotestis induction and/or feminised sexual characteristics. Although reproductive success may be impaired in some cases, implications for fish populations are still unclear. Suspected causative contaminants include natural oestrogenic substances and synthetic oestrogen mimics. The majority of the oestrogenic activity is adsorbed to sediments, and routes of exposure may include benthic food chain transfer. Some natural androgenic substances are also being discharged to estuaries, but their activity appears low.

The potential of the three-spined stickleback (Gasterosteus aculeatus L.) as a combined biomarker for oestrogens and androgens in European waters

The majority of endocrine disruption studies in Europe have been on non-indigenous species (some of them tropical!)--and none of which has traits that make them suitable for the detection of androgenic compounds. To overcome these problems, we have been developing the stickleback as a model biomarker for testing the effect of endocrine disrupters in European waters. Its advantages are: it is the only fish with a quantifiable in vivo androgen and anti-androgen endpoint (the production of the glue protein, spiggin, by the kidney); it is the only fish in which it will be possible to simultaneously test oestrogenic and androgenic properties of compound; it has a genetic sex marker; it is found in all EU countries; it survives and breeds in both seawater and freshwater; it is extremely robust and can be readily deployed in situ; it displays a variety of pronounced reproductive behaviours; it has a simple and short life cycle, low fecundity and high egg/fry survival rates.

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 transcriptomic and metabolomic responses in the Stickleback (Gasterosteus aculeatus) exposed to ethinyl-estradiol

An established three-spined stickleback (Gasterosteus aculeatus) cDNA array was expanded to 14,496 probes with the addition of hepatic clones derived from subtractive and normalized libraries from control males and males exposed to model toxicants. Microarrays and one-dimensional (1)H nuclear magnetic resonance (NMR) spectroscopy, together with individual protein and gene biomarkers were employed to investigate the hepatic responses of the stickleback to ethinyl-estradiol (EE(2)) exposure. Male fish were exposed via the water to EE(2), including environmentally relevant concentrations (0.1-100ng/l) for 4 days, and hepatic transcript and metabolite profiles, kidney spiggin protein and serum vitellogenin concentrations were determined in comparison to controls. EE(2) exposure did not significantly affect spiggin concentration but significantly induced serum vitellogenin protein at the threshold concentration of 32ng/l. (1)H NMR coupled with robust univariate testing revealed only limited changes, but these did support the predicted modulation of the amino acid profile by transcriptomics. Transcriptional induction was found for hepatic vitellogenins and choriogenins as expected, together with a range of other EE(2)-responsive genes. Choriogenins showed the more sensitive responses with statistically significant induction at 10ng/l. Real-time polymerase chain reaction (PCR) confirmed transcriptional induction of these genes. Phosvitinless vitellogenin C transcripts were highly expressed and represent a major form of the egg yolk precursors, and this is in contrast to other fish species where it is a minor component of vitellogenic transcripts. Differences in inducibility between the vitellogenins and choriogenins appear to be in accordance with the sequential formation of chorion and yolk during oogenesis in fish.

Towards a System Level Understanding of Non-Model Organisms Sampled from the Environment: A Network Biology Approach

The acquisition and analysis of datasets including multi-level omics and physiology from non-model species, sampled from field populations, is a formidable challenge, which so far has prevented the application of systems biology approaches. If successful, these could contribute enormously to improving our understanding of how populations of living organisms adapt to environmental stressors relating to, for example, pollution and climate. Here we describe the first application of a network inference approach integrating transcriptional, metabolic and phenotypic information representative of wild populations of the European flounder fish, sampled at seven estuarine locations in northern Europe with different degrees and profiles of chemical contaminants. We identified network modules, whose activity was predictive of environmental exposure and represented a link between molecular and morphometric indices. These sub-networks represented both known and candidate novel adverse outcome pathways representative of several aspects of human liver pathophysiology such as liver hyperplasia, fibrosis, and hepatocellular carcinoma. At the molecular level these pathways were linked to TNF alpha, TGF beta, PDGF, AGT and VEGF signalling. More generally, this pioneering study has important implications as it can be applied to model molecular mechanisms of compensatory adaptation to a wide range of scenarios in wild populations.

Detection of the anti-androgenic effect of endocrine disrupting environmental contaminants using in vivo and in vitro assays in the three-spined stickleback

We have previously developed a novel in vitro assay that utilises cultures of primed female stickleback kidney cells for the screening of potential androgenic and anti-androgenic environmental contaminants. Stickleback kidney cells are natural targets for steroid hormones and are able to produce a protein, spiggin, in response to androgenic stimulation. We undertook a combined in vivo/in vitro study where we used the magnitude of spiggin production as an endpoint to test the anti-androgenic properties of the pharmaceutical androgen antagonist flutamide and three environmental contaminants: the organophosphate insecticide fenitrothion, the urea-based herbicide linuron and the fungicide vinclozolin. In vitro, kidney cells were exposed to a range of concentrations [from 10(-14) M (2.5 pg/L) up to 10(-6) M (280 microg/L)] of the test compounds alone for determining agonist activities, or together with 10(-8) M (3 microg/L) dihydrotestosterone (DHT) for determining antagonist activities. An in vivo flow-through aquarium-based study was carried out in parallel. Female sticklebacks were exposed to a range of concentrations of the same chemicals alone or in combination with DHT (5 microg/L) for 21 days. All of the compounds significantly inhibited DHT-induced spiggin production in a concentration-dependent manner in both the in vitro (FN > or = FL > or = LN > VZ) and in vivo (FN > FL > or = VZ > LN) assays. Fenitrothion and flutamide inhibited spiggin production in vitro at a concentration as low as 10(-12) M (P < 0.05), while linuron and vinclozolin inhibited DHT-induced spiggin production at concentrations of 10(-10) M (P < 0.05) and 10(-6) M (P < 0.001) respectively. Similarly, fenitrothion and flutamide were the most potent chemicals in vivo and significantly reduced DHT-induced spiggin production at a concentration of 10 microg/L and 25 microg/L respectively (P < 0.01). Both linuron and vinclozolin induced a significant decrease in DHT-induced spiggin production at a concentration of 100 microg/L when tested in vivo. In addition, kidney cell primary culture was used to test the (anti-)androgenic effects of the major environmental contaminants: oestradiol (E2), nonylphenol (NP) and bisphenol A (BPA) for the first time in teleosts. We observed that these compounds were able to significantly inhibit spiggin production at high doses (E2: 270 microg/L; NP: 2.2 microg/L; BPA: 2.3 microg/L). When tested in the absence of DHT, none of the compounds showed a significant agonistic activity in either in vivo or in vitro assays. Overall, our data further demonstrate that kidney cell primary culture is a reliable and a sensitive screening tool for the detection of (anti-)androgenic compounds. In addition, our study represents the first attempt to develop a combined in vivo/in vitro screening strategy for assessing the effects of (anti-)androgenic endocrine disrupters.

The model anti-androgen flutamide suppresses the expression of typical male stickleback reproductive behaviour

Over the past 15 years considerable attention has been given to the presence in the environment of endocrine disrupting chemicals (EDCs) that may have harmful effects on organisms. Specific test guidelines for the detection of EDCs used for short-term fish screening assays have been developed by the Organisation for Economic Cooperation and Development (OECD). Compared to the core species used in the OECD guidelines, the three-spined stickleback (Gasterosteus aculeatus) has an additional and unique endpoint for (anti-)androgenic substances through the androgen-dependent glue protein (spiggin) used in the nest building. Here we describe a specific behavioural assay that was developed in parallel to the OECD protocol, utilising unique behavioural features of sticklebacks. In the assay, a photoperiod of 16L:8D (light:dark) and a temperature of 17+/-1 degrees C was used to induce breeding in quiescent male sticklebacks that were simultaneously exposed for a 21-day period to the mammalian anti-androgen flutamide (FL) at 100, 500 and 1000 microg/l (plus a water control). Spiggin production and the reproductive behaviour (nest building and courtship) of male sticklebacks were the main measured endpoints. The control fish entered an active breeding cycle including nest building and courtship behaviours as expected due to the stimulating temperature and photoperiodic conditions. The FL-exposed males showed significantly lower spiggin levels at 500 and 1000 microg/l. In addition, there was a significant decrease in the number of nests built by the FL-treated males at 100 microg/l with no nest built at 500 and 1000 microg/l. Finally, FL affected the courtship behaviour of the males with a significant reduction of the number of zigzags towards the female. When the breeding status of the stickleback males is controlled, the behavioural assay developed here is a suitable tool for the detection of androgen antagonists.