Elsevier

Aquatic Toxicology

Volume 90, Issue 2, 11 November 2008, Pages 83-91
Aquatic Toxicology

Transcriptomic responses of European flounder (Platichthys flesus) to model toxicants

https://doi.org/10.1016/j.aquatox.2008.07.019Get rights and content

Abstract

The temporal transcriptomic responses in liver of Platichthys flesus to model environmental pollutants were studied over a 16-day time span after intraperitoneal injection with cadmium chloride (50 μg/kg in saline), 3-methylcholanthrene (25 mg/kg in olive oil), Aroclor 1254 (50 mg/kg in olive oil), tert-butyl-hydroperoxide (5 mg/kg in saline), Lindane (25 mg/kg in olive oil), perfluoro-octanoic acid (100 mg/kg in olive oil) and their vehicles, olive oil (1 ml/kg) or saline (0.9%). Statistical, gene ontology and supervised analysis clearly demonstrated the progression from acute effects, biological responses to and recovery from the treatments. Key biological processes disturbed by the individual treatments were characterised by gene ontology analyses and individual toxicant-responsive genes and pathways were identified by supervised analyses. Responses to the polyaromatic and chlorinated aromatic compounds showed a degree of commonality but were distinguishable and they were clearly segregated from the responses to the pro-oxidants cadmium and the organic hydroperoxide, as well as from the peroxisomal proliferator, perfluoro-octanoic acid. This study demonstrated the utility of the microarray technique in the identification of toxicant-responsive genes and in discrimination between modes of toxicant action.

Introduction

Traditionally, many useful biomarkers of pollutant exposure in the environment have centered on the induction of individual stress-responsive genes, for example cytochrome P450 1A (CYP1A) in response to polycyclic aromatic hydrocarbons, metallothionein in response to heavy metals and vitellogenein in response to estrogenic compounds. Microarray technology offers an opportunity to gain a more comprehensive assessment of the health status of an organism through an understanding of the functional pathways that are responding to pollutant exposure and to identify new biomarkers. Through analysis of transcriptomics it has been demonstrated in vivo and in vitro that a profile of biological response to toxicants can be derived (Waring et al., 2001, Bartosiewicz et al., 2001, Hamadeh et al., 2002, McMillian et al., 2004), which can assist in the determination of the modes of action of chemicals on biological systems. Consequently this approach has the potential to aid risk assessment of chemicals and to assess the impact of chemical pollutants on organisms in the wild (Ankley et al., 2006). One distinct advantage of any of the “omic” technologies in this respect is that the assessment does not rely on the use of pre-determined end points, thus they have the potential to provide informative data on novel modes of action, particularly when used to assess the dose- and time-dependency of responses (Hamadeh et al., 2002, Moggs, 2005). The report by Pennie et al. (2004) suggests that biological pathways determined by transcriptomics are sufficiently robust to allow an insight into mechanisms of toxicity and that the initial uncertainty about oversensitivity may be unfounded. Whether many of these chemicals display similar modes of action in fish to those found in mammals has yet to be confirmed and a transcriptomic approach may therefore provide the required additional insight.

We have developed a cDNA microarray with 12,700 clones from the European flounder (Platichthys flesus) (the GENIPOL array, described in detail by Diab et al., 2008) and demonstrated the transcriptomic changes and biological pathways altered in the liver following exposure to cadmium (Williams et al., 2006), estradiol (Williams et al., 2007) and bacterial infection (Diab et al., 2008). We have now extended this approach to assess transcriptional responses following exposure to a range of different classes of inorganic and organic toxicants, thereby to test the ability to discriminate between different modes of action. In fish, this approach has been applied to carp, Cyprinus carpio (Moens et al., 2006) and the rainbow trout, Oncorhynchus mykiss (Koskinen et al., 2004, Hook et al., 2006, Tilton et al., 2006, Finne et al., 2007). These studies have allowed the identification of unique expression profiles elicited by particular toxicant treatments linking gene expression profiles and mechanisms of toxicity. Information of this nature is valuable in the context of screening novel chemicals for potential toxicity in fish. It is also important for the future development of systems to determine the holistic biological responses of fish exposed to mixtures of pollutants in the aquatic environment, the determination of classes of chemicals to which fish may be exposed and the development of predictive biomarkers. The choice of the European flounder for our studies is justified on the basis that this flatfish is commonly found in north-west European estuarine habitats in which it is frequently exposed to a range of pollutants in the sediment via its diet of benthic invertebrates (Kirby et al., 2004). It is a key sentinel species for biological effects monitoring in statutory monitoring programmes such as the OSPAR Joint Assessment and Monitoring Programme (OSPAR report, 1997). The liver was chosen as the target organ for this study as it is the major site of phase I and II metabolism and thus the tissue most frequently used in fish biomonitoring studies. Our microarray was derived, in the main, from hepatic cDNA libraries, so although it would be useful in assessing effects of toxicants on other organs, tissue-specific gene expression might not be captured.

In this study we have investigated responses in the liver of the European flounder following single exposures to a range of toxicants and studied the temporal changes in gene expression. In this way, not only did we determine the initial responses but we also gained information on the delayed responses and compared these profiles between the different chemical exposures.

Section snippets

Chemical treatments of fish

P. flesus were obtained as previously reported (Williams et al., 2006). At 2–3 months post-hatch, juveniles were transferred to a recirculating sea water system at Stirling University, Scotland and were on-grown at 11 °C, salinity 32 ppt. Sexually immature (macroscopically undifferentiated or male) fish of 75–100 g were treated by intraperitoneal injection with cadmium chloride (Cd, a hepatotoxic and carcinogenic heavy metal, 50 μg/kg in saline), 3-methylcholanthrene (3-MC, a planar polycyclic

Overview of results

Following dosage by intraperitoneal injection with any of the six chemicals or their carriers and subsequent starvation over the subsequent 16-day time course, there were no fatalities, nor significant effects on condition factor or liver somatic indices at the dosages used (p < 0.05, ANOVA, data not shown). Omission of food was consistent with the controls and overcame the potential for variability in gene expression related to effects of treatments on dietary intake. Determination of gene

The heavy metal, Cd

A mechanistic evaluation of response to cadmium was made in our previous report (Williams et al., 2006). Briefly, the initial toxic response invoked by Cd treatment was molecular damage and induction of apoptosis, with oxidative stress being the major and more sustained response. Metallothionein expression displayed an early induction whilst there was an early repression in immune function.

The organic hydroperoxide, tBHP

Overall the responses to tBHP were indicative of its character as an oxidative stressor. These responses

Conclusions

This study demonstrates that the key biological processes disrupted by toxicants involving different modes of action can be identified by transcriptomics with a non-model species. The data permit discrimination between classes of toxicants and discovery of novel biomarkers and the strategy is valuable in defining a general stress response which is of particular utility in determining the overall impact of toxicant mixtures upon an exposed organism. A question remains as to how responses to

Acknowledgements

This work was funded by EU ‘GENIPOL’ grant EKV-2001-0057 and UK NERC Post-genomic and Proteomic programme grant NE/C507661/1, Birmingham functional genomics facilities by BBSRC grant 6/JIF 13209 and bioinformatics by MRC infrastructure grant G.4500017. We thank Mr. A. Jones, and Dr. L. Klovrza for assistance.

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