tissue: liver developmental stage: juvenile treatment: control days of exposure: 7
Treatment protocol
Turbots were exposed to styrene for 7 days. Then fish were maintained for another 7 days in clean water for the recovery. The styrene exposure system was built in order to perform a controlled exposure to the chemical in a realistic way, simulating the possible concentration present in the sea surface after a styrene spill. The experimental system was composed of a mixing tank connected to an exposure tank and a degassing column. In the mixing tank, which had a capacity of 316 L, a slick of styrene was placed in contact with the aqueous phase and dissolved slowly. Then, contaminated water containing the dissolved fraction of the slick was pumped to the exposure tank (300 L) where turbots were maintained. Styrene disappeared rapidly from water and, therefore, the concentration remaining in the exposure tank would be similar to that appearing in seawater immediately after a spill. The exposure tank was connected to a system of waste water recuperation by overflow. During the experimentation, a water flow of 1.3 L min−1 was pumped and sent on a degassing column before being directed to the mixing tank. A second circuit ensured a renewal of fresh sea water to maintain the nitrate and nitrite concentrations stable. In addition, the oxygen saturation of water was maintained into this tank by a compressor, which injected air via diffuser. The degassing column favored the gaseous exchange, allowed water oxygenation by percolation on glass beads and allowed elimination of dissolved carbon dioxide produced by organisms (carbon dioxide transfer from water to air). During the experimentation, both mixing and exposure tanks were kept covered to avoid cross contamination from the styrene exposure tank to the control tank. The concentration of styrene was monitored in seawater during the experiment. Fish samples were collected before the exposure (T0), after 3 and 7 days of exposure and after 7 days of recovery in clean water. Muscle tissues of 10 control and 10 styrene-exposed turbots were collected after 7 days of exposure to determine the level of styrene in fish. Styrene concentration was measured in muscle because xenobiotic metabolism in this organ is much slower than in liver and, thus, styrene accumulation in muscle could better reflect environmental exposure. Livers of 6-7 turbots per experimental group at each sampling time were dissected out, immersed individually in RNA later® and frozen in liquid nitrogen for transcription studies. Livers, gonads and blood samples of 12 turbots per experimental group were collected and processed individually at each sampling time for liver AOX activity, plasma vitellogenin levels and histopathological analysis of liver and gonad. For lysosomal membrane stability test a small piece of the liver obtained from 5 fish per experimental group were rapidly frozen in liquid nitrogen and stored at −80 ◦C until sectioning. Additional information in Ruiz et al, 2012.
Growth protocol
Juvenile turbots, S. maximus (n = 500; weight 358 ± 93 g, length 28 ± 2 cm, mean ± SD) were purchased from a fish farm (France Turbot, Tredarzec, France). Before starting the experiment, they were acclimatized for 20 days in two laboratory tanks of 1000 L (control tank and treatment tank). They were fed daily with dried pellets (Aquaculture food Le Gouessant®, Lamballe, France, 4.5 mm diameter, total protein 54% of dry matter and crude fat 12% of dry matter). Running seawater was supplied at a flow rate of 5 L min−1 in both tanks. Water was taken from Sea of Iroise, France, and sterilized by means of UV-rays and filters system. Experiments were performed in the facilities of Cedre (Brest, France). The light regime was set according to the season: 14 h light, 10 h dark. Water salinity (35–36‰), water pH (7.98 ± 0.06), oxygen concentration (228 ± 18 μmol L−1) and sea water temperature (16 ± 2 °C) were measured daily. During the experiment, turbots were fed twice per day with dried pellets. Additional information in Ruiz et al, 2012.
Extracted molecule
total RNA
Extraction protocol
Extraction method: Trizol (Invitrogen) and further column purification (RNeasy Mini kit, QIAGEN)
Label
Cy-3-CTP
Label protocol
1st step: double stranded cDNA synthesis using MMLV-RT and Oligo dT-T7Promoter primer; 2nd Step: In vitro transcription and Cy-3 labeling of ds cDNA using the antisense T7 Promoter Primer,T7 RNA Polymerase and Cy-3-CTP; QuickAmp Labeling Kit one-color (Cy3-CTP included); 200 ng of purified total RNA were used in the labeled cRNA synthesis reaction.; Sample F-L-1 was labeled twice as a control
Hybridization protocol
600 ng of labeled cRNA were mixed with fragmentation buffer and blocking agent and fragmented by incubation at 60ºC for 30 minutes following protocol.; They were mixed with 2X GEX Hybridization buffer HI-RPM, and hybridize at 65ºC for 17h 45 min. Different samples wererandomly hybridized to different slides and arrays to avoid signal intensity differences due to hybridization/washing artifacts, spatial differences, etc
Image Analysis with Feature Extraction Software (FE; version:9.5.3.1 ); quantile normalization; average of copied probes; Data statistical analysis made in TIGR MultiExperiment Viewer (MEV) : MeV 4.0
HEPATIC GENE EXPRESSION PATTERNS IN JUVENILE TURBOTS (Scophthalmus maximus) AFTER EXPOSURE TO STYRENE (PRAGMA, a pragmatic and integrated approach for the evaluation of environmental impact of oil and chemical spills at sea)
Data table header descriptions
ID_REF
VALUE
RNA normalised signal intensity (the average of intensities of copied probes has been presented)
