Molecular responses of European flounder (Platichthys flesus) chronically exposed to contaminated estuarine sediments.

Molecular responses to acute toxicant exposure can be effective biomarkers, however responses to chronic exposure are less well characterised. The aim of this study was to determine chronic molecular responses to environmental mixtures in a controlled laboratory setting, free from the additional variability encountered with environmental sampling of wild organisms. Flounder fish were exposed in mesocosms for seven months to a contaminated estuarine sediment made by mixing material from the Forth (high organics) and Tyne (high metals and tributyltin) estuaries (FT) or a reference sediment from the Ythan estuary (Y). Chemical analyses demonstrated that FT sediment contained significantly higher concentrations of key environmental pollutants (including polycyclic aromatic hydrocarbons (PAHs), chlorinated biphenyls and heavy metals) than Y sediment, but that chronically exposed flounder showed a lack of differential accumulation of contaminants, including heavy metals. Biliary 1-hydroxypyrene concentration and erythrocyte DNA damage increased in FT-exposed fish. Transcriptomic and (1)H NMR metabolomic analyses of liver tissues detected small but statistically significant alterations between fish exposed to different sediments. These highlighted perturbance of immune response and apoptotic pathways, but there was a lack of response from traditional biomarker genes. Gene-chemical association annotation enrichment analyses suggested that polycyclic aromatic hydrocarbons were a major class of toxicants affecting the molecular responses of the exposed fish. This demonstrated that molecular responses of sentinel organisms can be detected after chronic mixed toxicant exposure and that these can be informative of key components of the mixture.

Transcriptomic responses of European flounder (Platichthys flesus) liver to a brominated flame retardant mixture.

Male European flounder (Platichthys flesus) were exposed to a technical mixture of brominated diphenyl ethers (PDBEs, DE-71, Pentamix) that had been purified to remove contaminating dioxins. Controls were exposed to carrier solvent alone. Fish were exposed to decadally increasing concentrations of Pentamix via both sediment and spiked food. The GENIPOL P. flesus cDNA microarray, differentially expressed gene profiling (DEG) and quantitative PCR were employed to detect hepatic transcriptional differences between exposed fish and controls. Gene transcriptional changes were more sensitive to Pentamix exposure than biomarkers measured previously. Pentamix exposure induced transcripts coding for enzymes of xenobiotic metabolism (CYP1A, aldo-keto reductases) and elicited endocrine disruption (vitellogenin and thyroid hormone receptor alpha), with effects on CYP1A and VTG occurring at the highest exposure. Ontology analysis clearly showed dose-responsive changes indicative of oxidative stress, induction of mitochondrial dysfunction, and apoptosis. We conclude that exposure to PBDEs in both sediment and food has a significant adverse effect on a broad range of crucial biochemical processes in the livers of this widely distributed estuarine fish species, the flounder.

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.

Hepatic gene expression in flounder chronically exposed to multiply polluted estuarine sediment: Absence of classical exposure 'biomarker' signals and induction of inflammatory, innate immune and apoptotic pathways.

The effects of chronic long-term exposure to multiply polluted environments on fish are not well understood, but environmental surveys suggest that such exposure may cause a variety of pathologies, including cancers. Transcriptomic profiling has recently been used to assess gene expression in European flounder (Platichthys flesus) living in several polluted and clean estuaries. However, the gene expression changes detected were not unequivocally elicited by pollution, most likely due to the confounding effects of natural estuarine ecosystem variables. In this study flounder from an uncontaminated estuary were held on clean or polluted sediments in mesocosms, allowing control of variables such as salinity, temperature, and diet. After 7 months flounder were removed from each mesocosm and hepatocytes prepared from fish exposed to clean or polluted sediments. The hepatocytes were treated with benzo(a)pyrene (BAP), estradiol (E2), copper, a mixture of these three, or with the vehicle DMSO. A flounder cDNA microarray was then used to measure hepatocyte transcript abundance after each treatment. The results show that long-term chronic exposure to a multiply polluted sediment causes increases in the expression of mRNAs coding for proteins of the endogenous apoptotic programme, of innate immunity and inflammation. Contrary to expectation, the expression of mRNAs which are commonly used as biomarkers of environmental exposure to particular contaminants were not changed, or were changed contrary to expectation. However, acute treatment of hepatocytes from flounder from both clean and polluted sediments with BAP or E2 caused the expected changes in the expression of these biomarkers. Thus transcriptomic analysis of flounder exposed long-term to chronic pollution causes a different pattern of gene expression than in fish acutely treated with single chemicals, and reveals novel potential biomarkers of environmental contaminant exposure. These novel biomarkers include Diablo, a gene involved in apoptotic pathways and highly differentially regulated by both chronic and acute exposure to multiple pollutants.

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

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 microg/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 (25mg/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.

Protein expression by Aeromonas hydrophila during growth in vitro and in vivo.

Expression of Aeromonas hydrophila cellular and extracellular products (ECPs) was examined following culture of the bacterium in vitro, in Tryptic Soy Broth (TSB), and in vivo, in dialysis tubing placed within the peritoneal cavity of common carp (Cyprinus carpio L.). Whole cell (WC), outer membrane proteins (OMPs) and ECP components of the bacteria were analysed by 1 dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis (1D SDS-PAGE). Additionally, 2D SDS-PAGE was used to analyse WC preparations. The aim of the study was to identify unique and common proteins up-regulated in vivo. Unique bands were seen in the 1D gels at 58 and 55 kDa for WC and OMP preparations, respectively, for all the four virulent and two avirulent isolates cultured in vivo. Bands of increased intensity were also observed at 70, 55, 50 and 25 kDa with WC preparations for all virulent isolates cultured in vivo. Analysis of WC by 2D SDS-PAGE revealed that bacteria cultured in vivo expressed a number of unique spots, mostly between 30 and 80 kDa with pI values ranging from 5.0 to 6.0. The unique proteins identified in vivo may be involved in the virulence of the bacterium and their potential as vaccine candidates is currently being investigated.

Gene expression responses of European flounder (Platichthys flesus) to 17-beta estradiol.

Male European flounder (Platichthys flesus) were intraperitoneally injected with 10mg/kg 17-beta estradiol and tissues taken from individuals over a timecourse of 16 days. The GENIPOL P. flesus cDNA microarray was employed to detect hepatic gene expression differences between fish treated with estradiol and saline controls. Known biomarkers of estrogen exposure, choriogenin L and vitellogenins, showed sustained induction over the time-course. Among 175 identified clones showing sustained statistically significant induction or repression, those associated with the Gene Ontology terms mitochondria, amino acid synthesis, ubiquitination and apoptosis were included amongst those induced while those associated with immune function, electron transport, cell signalling and protein phosphorylation were repressed. Thus, we show the gene expression response of an environmentally relevant fish species to a high dose of an estrogenic endocrine disruptor and also report the sequencing of a further 2121 flounder ESTs.

Development of the GENIPOL European flounder (Platichthys flesus) microarray and determination of temporal transcriptional responses to cadmium at low dose.

We have constructed a high density, 13 270-clone cDNA array for the sentinel fish species European flounder (Platichthys flesus), combining clones from suppressive subtractive hybridization and a liver cDNA library; DNA sequences of 5211 clones were determined. Fish were treated by single intraperitoneal injection with 50 micrograms cadmium chloride per kilogram body weight, a dose relevant to environmental exposures, and hepatic gene expression changes were determined at 1, 2, 4, 8, and 16 days postinjection in comparison to saline-treated controls. Gene expression responses were confirmed by real-time reverse transcription polymerase chain reaction (RT-PCR). Blast2GO gene ontology analysis highlighted a general induction of the unfolded protein response, response to oxidative stress, protein synthesis, transport, and degradation pathways, while apoptosis, cell cycle, cytoskeleton, and cytokine genes were also affected. Transcript levels of cytochrome P450 1A (CYP1A) were repressed and vitellogenin altered, real-time PCR showed induction of metallothionein. We thus describe the establishment of a useful resource for ecotoxicogenomics and the determination of the temporal molecular responses to cadmium, a prototypical heavy metal pollutant.

Benzothiazole degradation by Rhodococcus pyridinovorans strain PA: evidence of a catechol 1,2-dioxygenase activity.

The pathway for biodegradation of benzothiazole (BT) and 2-hydroxybenzothiazole (OBT) by Rhodococcus pyridinovorans strain PA was studied in detail. The kinetics of biodegradation were monitored by in situ (1)H nuclear magnetic resonance (NMR) in parallel with reversed-phase high-performance liquid chromatography (HPLC). Successive oxidations from BT to OBT and then from OBT to dihydroxybenzothiazole were observed. Further insight was obtained by using a mutant strain with impaired ability to grow on BT and OBT. The precise structure of another intermediate was determined by in situ two-dimensional (1)H-(13)C NMR and HPLC-electrospray ionization mass spectrometry; this intermediate was found to be a ring-opening product (a diacid structure). Detection of this metabolite, together with the results obtained by (1)H and (19)F NMR when cells were incubated with 3-fluorocatechol, demonstrated that a catechol 1,2-dioxygenase is involved in a pathway for biodegradation of BTs in this Rhodococcus strain. Our results show that catechol 1,2-dioxygenase and catechol 2,3-dioxygenase activities may both be involved in the biodegradation of BTs depending on the culture conditions.