Matthew B. Sanders

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.

Ocean acidification increases the toxicity of contaminated sediments

Ocean acidification (OA) may alter the behaviour of sediment-bound metals, modifying their bioavailability and thus toxicity. We provide the first experimental test of this hypothesis with the amphipod Corophium volutator. Amphipods were exposed to two test sediments, one with relatively high metals concentrations (Σmetals 239 mg kg(-1) ) and a reference sediment with lower contamination (Σmetals 82 mg kg(-1) ) under conditions that mimic current and projected conditions of OA (390-1140 μatm pCO2 ). Survival and DNA damage was measured in the amphipods, whereas the flux of labile metals was measured in the sediment and water column (WC) using Diffusive Gradients in Thin-films. The contaminated sediments became more acutely toxic to C. volutator under elevated pCO2 (1140 μatm). There was also a 2.7-fold increase in DNA damage in amphipods exposed to the contaminated sediment at 750 μatm pCO2 , as well as increased DNA damage in organisms exposed to the reference sediment, but only at 1140 μatm pCO2 . The projected pCO2 concentrations increased the flux of nickel and zinc to labile states in the WC and pore water. However, the increase in metal flux at elevated pCO2 was equal between the reference and contaminated sediments or, occasionally, greater from reference sediments. Hence, the toxicological interaction between OA and contaminants could not be explained by e ffects of pH on metal speciation. We propose that the additive physiological effects of OA and contaminants will be more important than changes in metal speciation in determining the responses of benthos to contaminated sediments under OA. Our data demonstrate clear potential for near-future OA to increase the susceptibility of benthic ecosystems to contaminants. Environmental policy should consider contaminants within the context of changing environmental conditions. Specifically, sediment metals guidelines may need to be reevaluated to afford appropriate environmental protection under future conditions of OA.

Meeting Report: Risk Assessment of Tamiflu Use Under Pandemic Conditions

On 3 October 2007, 40 participants with diverse expertise attended the workshop Tamiflu and the Environment: Implications of Use under Pandemic Conditions to assess the potential human health impact and environmental hazards associated with use of Tamiflu during an influenza pandemic. Based on the identification and risk-ranking of knowledge gaps, the consensus was that oseltamivir ethylester-phosphate (OE-P) and oseltamivir carboxylate (OC) were unlikely to pose an ecotoxicologic hazard to freshwater organisms. OC in river water might hasten the generation of OC-resistance in wildfowl, but this possibility seems less likely than the potential disruption that could be posed by OC and other pharmaceuticals to the operation of sewage treatment plants. The work-group members agreed on the following research priorities: a) available data on the ecotoxicology of OE-P and OC should be published; b) risk should be assessed for OC-contaminated river water generating OC-resistant viruses in wildfowl; c) sewage treatment plant functioning due to microbial inhibition by neuraminidase inhibitors and other antimicrobials used during a pandemic should be investigated; and d) realistic worst-case exposure scenarios should be developed. Additional modeling would be useful to identify localized areas within river catchments that might be prone to high pharmaceutical concentrations in sewage treatment plant effluent. Ongoing seasonal use of Tamiflu in Japan offers opportunities for researchers to assess how much OC enters and persists in the aquatic environment.

Effects of water accommodated fractions of crude oils and diesel on a suite of biomarkers in Atlantic cod (Gadus morhua)

The aim of this study was to characterize concentration- and time-dependent responses in juvenile Atlantic cod (Gadus morhua) following exposure for one and three weeks to the water-soluble fraction (WAF) of three weathered oils: Arabian Light crude oil (ALC), North Sea crude oil (NSC) and ship-diesel. The sum of polycyclic aromatic hydrocarbons (PAH) in water was highest after one week of exposure and within environmentally relevant concentrations. PAH metabolites in bile confirmed exposure to and uptake of PAHs. Hepatic cytochrome P450 1A (CYP1A) gene expression (mRNA quantification) increased dramatically following exposure to all three oil types (fold-change up to 165) and there was a time lag between gene and protein expression. Hepatic CYP1A protein concentration and ethoxyresorufin-O-deethylase (EROD) activity were more variable among individuals and treatments than gene expression. EROD activity in liver and gills increased in fish exposed to WAF from the two crude oils, but not in fish exposed to WAF from diesel. Exposure to diesel appeared to induce oxidative stress to a greater extent than exposure to crude oils. Other biomarkers (glutathione S-transferases, acetylcholine esterase, vitellogenin) did not appear to respond to the exposure and hence did not discriminate among oils. Biomarker responses in cod after exposure to weathered crude oils and diesel suggested that the CYP1A system and oxidative stress markers have the highest potential for discriminating among different oil types and to monitor the environmental consequences of spills.

Intercalibration exercise using a stickleback endocrine disrupter screening assay

The Organisation for Economic Cooperation and Development (OECD) is currently validating a short-term fish screening protocol for endocrine disrupters (estrogens, androgens, and their antagonists and aromatase inhibitors), using three core species: fathead minnow, Japanese medaka, and zebrafish. The main endpoints proposed for the first phase of validation of the screen are vitellogenin (VTG) concentration, gross morphology (secondary sexual characteristics and gonado-somatic index), and gonadal histopathology. A similar protocol is concurrently being developed in the United Kingdom using the three-spined stickleback, with identical endpoints to those for the core species and, in addition, a unique androgen-specific endpoint in the form of spiggin (glue protein) induction. To assess the suitability of this species for inclusion in the OECD protocol alongside the core species, an intercalibration was conducted using 17beta-estradiol (a natural estrogen) and trenbolone (a synthetic androgen), thus mimicking a previous intercalibration with the core species. All three participating laboratories detected statistically significant increases in VTG in males after 14 d exposure to nominal concentrations of 100 ng/L 17beta-estradiol and statistically significant increases in spiggin in females after 14 d exposure to nominal concentrations of 5,000 ng/L trenbolone. The stickleback screen is reliable, possessing both relevant and reproducible endpoints for the detection of potent estrogens and androgens. Further work is underway to assess the relevance and suitability of the screen for weakly acting estrogens, anti-androgens, and aromatase inhibitors.

Juvenile king scallop, Pecten maximus, is potentially tolerant to low levels of ocean acidification when food is unrestricted.

The decline in ocean water pH and changes in carbonate saturation states through anthropogenically mediated increases in atmospheric CO2 levels may pose a hazard to marine organisms. This may be particularly acute for those species reliant on calcareous structures like shells and exoskeletons. This is of particular concern in the case of valuable commercially exploited species such as the king scallop, Pecten maximus. In this study we investigated the effects on oxygen consumption, clearance rates and cellular turnover in juvenile P . maximus following 3 months laboratory exposure to four pCO2 treatments (290, 380, 750 and 1140 µatm). None of the exposure levels were found to have significant effect on the clearance rates, respiration rates, condition index or cellular turnover (RNA: DNA) of individuals. While it is clear that some life stages of marine bivalves appear susceptible to future levels of ocean acidification, particularly under food limiting conditions, the results from this study suggest that where food is in abundance, bivalves like juvenile P . maximus may display a tolerance to limited changes in seawater chemistry.

Construction of subtracted EST and normalised cDNA libraries from liver of chemical-exposed three-spined stickleback (Gasterosteus aculeatus) containing pollutant-responsive genes as a resource for transcriptome analysis.

The three-spined stickleback (Gasterosteus aculeatus) is ideally suited to laboratory studies, while its wide distribution in the northern hemisphere gives it great potential as a sentinel organism. In the setting of a UK-wide collaboration (Fish Toxicogenomics) we have developed a microarray for transcriptomic analysis of chemical responses in populations of G. aculeatus under laboratory and field conditions. Although several EST libraries are available for this species none are from chemical-exposed fish and thus unlikely to include a full set of pollutant-responsive genes. To harvest such transcripts cDNA libraries were produced from liver of chemical-exposed mature males. Two normalised full-length libraries were generated by different methods: (1) partial subtraction of polyA+ RNA against solid-phase cDNA using magnetic bead technology; (2) degradation of double stranded cDNA formed by abundant transcripts. To enrich for pollutant-responsive genes a subtracted EST library was also generated. For each library approximately 1.5K clones were sequenced and characterised using Blast2GO. All libraries contained pollutant-responsive transcripts not previously available while additionally the subtracted library was generally enriched approximately 1.2-10-fold for transcripts expected to be induced in response to the pollutants.

Exposure of sticklebacks (Gasterosteus aculeatus) to cadmium sulfide nanoparticles: biological effects and the importance of experimental design.

Effects of nanoparticles on aquatic organisms have been little studied to date and toxicological data are urgently needed for development of regulatory frameworks for these substances. Here, we report the findings of a study exposing sticklebacks to cadmium sulfide (CdS) as bulk material and quantum dots. Fish were exposed for 21 d in a flow through test system to 5, 50 or 500 microg l(-1) CdS nanoparticles (nCdS) coated in thiol terminated methyl polyethylene glycol (MPEG), bulk CdS or MPEG at 500 microg l(-1) (nominal concentrations). With the exception of the highest nCdS exposure, measured concentrations were approximately one order of magnitude below nominal. A single fish from each group (excluding MPEG) was examined using energy dispersive X-ray analysis (EDX) to localise cadmium, however, cadmium could not be detected in whole body sections. Elevated levels of oxidized glutathione were measured in the gills of fish exposed to 50 and 500 microg l(-1) nCdS. Induction of vitellogenin synthesis was not detected in any of the treatment groups. The number of males engaged in nest-building behaviour following exposure to 500 microg l(-1) nCdS was reduced and livers of 4/6 fish in the same treatment displayed hepatocellular nuclear pleomorphism. The results are discussed emphasising the fundamental importance of experimental design and the need to understand the behaviour of nanoparticles in the aqueous phase.

Genotoxic effects of CdS quantum dots and Ag2S nanoparticles in fish cell lines (RTG-2)

The increasing use of nanotechnologies will lead to significant releases of engineered nanoparticles into the aquatic environment, where their impact is still poorly characterized. In the present paper, the genotoxic and cytotoxic properties of CdS quantum dots (QDs) and silver sulphide (Ag2S) coated with methyl polyethylene glycol (M-PEG) were investigated in a rainbow trout cell line (RTG-2). The results showed that CdS QDs were highly cytotoxic at high concentrations (10 and 50μg/ml), and exhibited a concentration-dependent genotoxicity in the sub-toxic range (0.01-1μg/ml) after 24h exposure. Ag2S showed neither genotoxic nor cytotoxic effects.

Genotoxic effects of Ag2S and CdS nanoparticles in blue mussel (Mytilus edulis) haemocytes

The use of functionalised metal sulphide nanoparticles (NPs) for nanoremediation and biomedical application is rapidly increasing, which could lead to significant inputs into the marine environment. The potential impact of some NPs on marine organisms is still poorly understood. In the present paper the genotoxic potential of Ag2S and CdS NPs on Mytilus edulis haemocytes was assessed. MPEG-SH (thiol-terminated methyl polyethylene glycol), was used as capping agent to avoid NPs agglomeration. TEM analysis showed that the Ag2S NPs size was 13±7 nm, whereas CdS quantum dots had an average diameter of 4±1 nm. DNA integrity was evaluated by Comet assay following exposure to increasing concentration series (0.01–10 mg/L). Both silver and cadmium NPs showed genotoxic effects at the highest dose. MPEG-SH was also found to exert a weak genotoxic activity, suggesting that at least part of the genotoxic potential of functionalised NPs on mussel cells might be attributable to the capping agent. These results confirm the genotoxic potential of Ag2S NPs for mussel cells and demonstrated, for the first time, that CdS NPs is genotoxic in a marine organism.