Rachel C. Skirrow

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.

Toxicity of silver nanoparticles to rainbow trout: a toxicogenomic approach.

Silver (Ag) nanoparticles are used as antimicrobial adjuvant in various products such as clothes and medical devices where the release of nano-Ag could contaminate the environment and harm wildlife. The purpose of this study was to examine the sublethal effects of nano-Ag and dissolved Ag on Oncorhynchus mykiss rainbow trout. Hepatic Ag contents and changes in gene expression were monitored to provide insights on bioavailability and mode of action of both forms of silver. Fish were exposed to increasing concentrations (0.06, 0.6 and 6 μg L(-1)) of nano-Ag (20 nm) and silver nitrate (AgNO(3)) for 96 h at 15°C. A gene expression analysis was performed in the liver using a DNA microarray of 207 stress-related genes followed by a quantitative polymerase chain reaction on a selection of genes for validation. The biochemical markers consisted of the determination of labile zinc, metallothioneins, DNA strand breaks, lipid peroxidation (LPO) and vitellogenin-like proteins. The analysis of total Ag in the aquarium water revealed that nano-Ag was mostly aggregated, with 1% of the total Ag being dissolved. Nevertheless, hepatic Ag content was significantly increased in exposed fish. Indeed, dissolved Ag was significantly more bioavailable than nano-Ag only at the highest concentration with 38 ± 10 and 11 ± 3 ng Ag mg(-1) proteins for dissolved and nano-Ag respectively. Exposure to both forms of Ag led to significant changes in gene expression for 13% of tested gene targets. About 12% of genes responded specifically to nano-Ag, while 10% of total gene targets responded specifically to dissolved Ag. The levels of vitellogenin-like proteins and DNA strand breaks were significantly reduced by both forms of Ag, but DNA break levels were lower with nano-Ag and could not be explained by the presence of ionic Ag. Labile zinc and the oxidized fraction of metallothioneins were increased by both forms of Ag, but LPO was significantly induced by nano-Ag only. A discriminant function analysis revealed that the responses obtained by biochemical markers and a selection of ten target genes were able to discriminate completely (100%) the effects of both forms of Ag. Exposure to nano-Ag involved genes in inflammation and dissolved Ag involved oxidative stress and protein stability. Hence, the toxicity of Ag will differ depending on the presence of Ag nanoparticles and aggregates.

Immunocompetence and alterations in hepatic gene expression in rainbow trout exposed to CdS/CdTe quantum dots

Cadmium (Cd) tellurium quantum dots (QDs) are long-lived fluorescent nanocrystals that have the ability to produce potentially toxic reactive oxygen species at the surface of the nanoparticle and release toxic cadmium ions. The purpose of this study was to examine the sublethal effects of CdS/CdTe QDs and dissolved Cd on the immune system of rainbow trout. Changes in hepatic gene expression were also monitored to provide insight on the mode of action of both forms of cadmium. Oncorhynchus mykiss fish were exposed to increasing concentrations of CdS/CdTe or dissolved Cd (CdSO(4)) for 48 h at 15 degrees C. The anterior head kidney was analyzed for leukocyte count, viability and phagocytic activity. The livers were harvested and prepared for gene expression analysis using a DNA microarray comprised of 207 stress-related genes. An analysis of total Cd in the aquarium water revealed that the nominal concentrations corresponded well to the actual concentrations and that a small proportion (0.4%) of Cd in the QDs was found in the dissolved fraction. Exposure to QDs led to significant reductions in leukocyte counts, viability and both resting and active phagocytic activity. On a mass concentration basis, QDs were more potent than dissolved Cd in suppressing immunocompetence in rainbow trout. The analysis revealed that both forms of Cd were strong inducers of metallothionein and CP2K1 gene expressions, which are respectively involved in metal detoxification processes and xenobiotic transformation/inflammation conditions. The analysis revealed different modes of action for each form of Cd. For QDs, 25 genes specific to QDs and related to the immune endpoints were found. The genes were involved in inflammation, xenobiotic biotransformation and endocrine system (including the induction of vitellogenin and its receptor). The effect for dissolved Cd was narrower than for QDs, with 9 genes specific to dissolved Cd being strongly correlated with the observed effects on immunocompetence. The involved genes were binding and transport of various solutes (urea, sodium, potassium) and the complement system. The present study revealed that each form of Cd produced a different pattern of gene expression and lowered fish immunocompetence.

Triclosan exposure alters postembryonic development in a Pacific tree frog (Pseudacris regilla) Amphibian Metamorphosis Assay (TREEMA)

The Amphibian Metamorphosis Assay (AMA), developed for Xenopus laevis, is designed to identify chemicals that disrupt thyroid hormone (TH)-mediated biological processes. We adapted the AMA for use on an ecologically-relevant North American species, the Pacific tree frog (Pseudacris regilla), and applied molecular endpoints to evaluate the effects of the antibacterial agent, triclosan (TCS). Premetamorphic (Gosner stage 26-28) tadpoles were immersed for 21 days in solvent control, 1.5 μg/L thyroxine (T(4)), 0.3, 3 and 30 μg/L (nominal) TCS, or combined T(4)/TCS treatments. Exposure effects were scored by morphometric (developmental stage, wet weight, and body, snout-vent and hindlimb lengths) and molecular (mRNA abundance using quantitative real time polymerase chain reaction) criteria. T(4) treatment alone accelerated development concomitant with altered levels of TH receptors α and β, proliferating cell nuclear antigen, and gelatinase B mRNAs in the brain and tail. We observed TCS-induced perturbations in all of the molecular and morphological endpoints indicating that TCS exposure disrupts coordination of postembryonic tadpole development. Clear alterations in molecular endpoints were evident at day 2 whereas the earliest morphological effects appeared at day 4 and were most evident at day 21. Although TCS alone (3 and 30 μg/L) was protective against tadpole mortality, this protection was lost in the presence of T(4). The Pacific tree frog is the most sensitive species examined to date displaying disruption of TH-mediated development by a common antimicrobial agent.

Evaluating the treatment of a synthetic wastewater containing a pharmaceutical and personal care product chemical cocktail: Compound removal efficiency and effects on juvenile rainbow trout

Pharmaceutical and personal care products (PPCPs) can evade degradation in sewage treatment plants (STPs) and can be chronically discharged into the environment, causing concern for aquatic organisms, wildlife, and humans that may be exposed to these bioactive chemicals. The ability of a common STP process, conventional activated sludge (CAS), to remove PPCPs (caffeine, di(2-ethylhexyl)phthalate, estrone, 17α-ethinylestradiol, ibuprofen, naproxen, 4-nonylphenol, tonalide, triclocarban and triclosan) from a synthetic wastewater was evaluated in the present study. The removal of individual PPCPs by the laboratory-scale CAS treatment plant ranged from 40 to 99.6%. While the efficiency of removal for some compounds was high, remaining quantities have the potential to affect aquatic organisms even at low concentrations. Juvenile rainbow trout (Oncorhynchus mykiss) were exposed to influent recreated model wastewater with methanol (IM, solvent control) or with PPCP cocktail (IC), or CAS-treated effluent wastewater with methanol (EM, treated control) or with PPCP cocktail (EC). Alterations in hepatic gene expression (evaluated using a quantitative nuclease protection plex assay) and plasma vitellogenin (VTG) protein concentrations occurred in exposed fish. Although there was partial PPCP removal by CAS treatment, the 20% lower VTG transcript levels and 83% lower plasma VTG protein concentration found in EC-exposed fish compared to IC-exposed fish were not statistically significant. Thus, estrogenic activity found in the influent was retained in the effluent even though typical percent removal levels were achieved raising the issue that greater reduction in contaminant load is required to address hormone active agents.

Effects of acute exposure to the non-steroidal anti-inflammatory drug ibuprofen on the developing North American Bullfrog (Rana catesbeiana) tadpole.

A variety of pharmaceutical chemicals can represent constituents of municipal effluent outflows that are dispersed into aquatic receiving environments worldwide. Increasingly, there is concern as to the potential of such bioactive substances to interact with wildlife species at sensitive life stages and affect their biology. Using a combination of DNA microarray, quantitative real-time polymerase chain reaction, and quantitative nuclease protection assays, we assessed the ability of sub-lethal and environmentally relevant concentrations of ibuprofen (IBF), a non-steroidal anti-inflammatory agent and prevalent environmental contaminant, to function as a disruptor of endocrine-mediated post-embryonic development of the frog. While the LC50 of IBF for pre-metamorphic Rana catesbeiana tadpoles is 41.5 mg/L (95% confidence interval: 32.3-53.5 mg/L), exposure to concentrations in the ppb range elicited molecular responses both in vivo and in organ culture. A nominal concentration of 15 μg/L IBF (actual = 13.7 μg/L) altered the abundance of 26 mRNA transcripts within the liver of exposed pre-metamorphic R. catesbeiana tadpoles within 6 d. IBF-treated animals demonstrated subsequent disruption of thyroid hormone-mediated reprogramming in the liver transcriptome affecting constituents of several metabolic, developmental, and signaling pathways. Cultured tadpole tail fin treated with IBF for 48 h also demonstrated altered mRNA levels at drug concentrations as low as 1.5 μg/L. These observations raise the possibility that IBF may alter the post-embryonic development of anuran species in freshwater environs, where IBF is a persistent or seasonal pollutant.

Minimally invasive transcriptome profiling in salmon: Detection of biological response in rainbow trout caudal fin following exposure to environmental chemical contaminants

An increasing number of anthropogenic chemicals have demonstrated potential for disruption of biological processes critical to normal growth and development of wildlife species. Both anadromous and freshwater salmon species are at risk of exposure to environmental chemical contaminants that may affect migratory behavior, environmental fitness, and reproductive success. A sensitive metric in determination of the presence and impact of such environmental chemical contaminants is through detection of changes in the status of gene transcript levels using a targeted quantitative real-time polymerase chain reaction assay. Ideally, the wildlife assessment strategy would incorporate conservation-centered non-lethal practices. Herein, we describe the development of such an assay for rainbow trout, Oncorhynchus mykiss, following an acute 96 h exposure to increasing concentrations of either 17α-ethinyl estradiol or cadmium. The estrogenic screen included measurement of mRNA encoding estrogen receptor α and β isoforms, vitellogenin, vitelline envelope protein γ, cytochrome p450 family 19 subfamily A, aryl hydrocarbon receptor, and the stress indicator, catalase. The metal exposure screen included evaluation of the latter two mRNA transcripts along with those encoding the metallothionein A and B isoforms. Exposure-dependent transcript abundance profiles were detected in both liver and caudal fin supporting the use of the caudal fin as a non-lethally obtained tissue source. The potential for both transcriptome profiling and genotypic sex determination from fin biopsy was extended, in principle, to field-captured Chinook salmon (Oncorhynchus tshawytscha).