POLLUTANTS:
The effects of a variety of prototypical toxicants and important environmental pollutants are being tested experimentally to establish a database of effects upon gene and protein expression which can be used diagnostically for these and other compounds with the same mode of action or pathological effect.

Protypical and pollutant compounds being investigated.

   

Polyaromatic hydrocarbons

3-MethylCholanthrene, Benzo(a)pyrene

Polyhalogentated aromatic hydrocarbons

Arochlor 1254, Dioxin, Lindane

Endocrine disruptive compounds,

17-b Estradiol, 17Methyltestosterone, 4-NonylPhenol, Bisphenol A, Organotins, DDE, DDT

Peroxisomal proliferators

Perfluoroctanoic acid

Heavy metals

Cadmium, Lead, Nickel

Oxidative stressors:

t-Butylhydroperoxide, Lindane

Immunosuppressants

Prednisolone, PCN, Dioxin

Stressors

Corticosterone, dexamethasone

Pesticides

lindane and others to be finalised

DNA damaging agents  MNNU
   

Environmental exposures:

Sediments and sediment extracts from various polluted areas, including:-
Clyde estuary, Haifa Bay, Mersey estuary, Rotterdam harbour, Swansea Docks, R. Tyne.

FLOUNDERS:

Flounders were obtained by trawl from a clean area of the Irish Sea and maintained in a flow through sea water system at the University of Liverpool, Port Erin Marine laboratory, Port Erin, Isle of Man, UK. Gametes were stripped from ripe animals and fish were artificially reared from 3 x 3 crosses of mixed gamete pools. After hatching, larvae were fed algae/rotifers from days 3-6, then Artemia salina from days 5-25 and weaned onto an artificial diet (Stirling prepared)on day 20 which was then substituted after metamorphosis by a commercial pelleted diet (Trou no.3). Animals were used experimentally from 8-18 months post hatch.

Control fish:

10 sexually mature females; 10 sexually mature males; 10 immature males

EXPERIMENTAL EXPOSURES.............................................In Vivo treatments:

Batches of 25-30 fish were exposed to pollutant compounds in vehicle (0.9% saline or olive oil, 1ml/kg) by intraperitoneal injection.

Toxicant

Supplier, code

Dose, vehicle

3-methylcholanthrene

Sigma M6501

25mg/kg in olive oil

Benzo(a)pyrene Sigma  

Arochlor 1254

ICI , direct gift

50mg/kg in olive oil

Perfluoro-octanoic acid

Fluka

100mg/kg in olive oil

t-butylhydroperoxide

Sigma B2633

5mg/kg in 0.9% saline

17-b Estradiol

Sigma E8875

10mg/kg in olive oil

17-a Methyltestosterone

Sigma M7252

10mg/kg in olive oil

Cadmium chloride

Sigma C3141

50mg/kg in 0.9% saline

PCN   10mg/kg
Lindane   25mg/kg
     

Fish were maintained at 12°C in aerated sea water (static, changed twice weekly) and sampled as below.

Sampling:

Day 0, 1, 2, 4, 8 200mg tissue(liver, kidney) homogenised in 2ml TriReagent (Sigma),

Stored overnight at -40°C then RNA extracted within 2days. RNA stored under ethanol at -85°C.

Day 0, 4, 8, 16 ca 1-2 mm3pieces of tissue placed in straws and plunged into liquid nitrogen.

Stored at -85°C. Shipped in liquid nitrogen for protein analysis. Remainder of tissue "snap frozen" in liquid nitrogen in 1ml cryovials and stored at -85°C. 5mm cubes sent for proteome analysis.

Partner site has Animal/ sample databases.

EXPERIMENTAL EXPOSURES ...............................................In vitro treatments:

Hepatocytes were isolated from male fish prepared as described in Morrison & George, (1985) Biochem. Pharmacol. 34, 3933-3938. They were cultured at 15°C in an atmosphere of 5% CO2 in Liebowitz L15 supplemented with 50u/ml penicillin, 50ug/ml streptomycin, 0.5mg/ml kanomycin, 50ug/ml gentomycin, 50ug/ml L-glutamine, 10% foetal calf serum, 1.25mg/ml NaHCO3, 0.26mg/ml CaCl2.

FIELD EXPOSURES AND COMPARISON WITH OTHER BIOLOGICAL EFFECTS MEASUREMENTS

Flounders have been sampled by trawling during October/December 2002 from 22 stations (20 male, 20 female fish per

station in 12 UK estuaries as shown in the map.

 

The St. Andrews Bay and Alde estuary sites are considered reference sites. The sites are UK NMMP sites where environmental and organismal chemistry, biology and toxicity testing are carried out and therefore represent well characterised environments for biological impact analysis.

 

Fish are being analysed for the ICES PAH suite (EROD, bile metabolites, DNA adducts, histopathology), endocrine disruption (plasma Vitellogenin) and metal effects (metallothionein induction) as part of the DEMECS program by the UK National marine monitoring program ecotoxicology quality control group and data will be submitted to ICES. Two aliquots of the same livers were taken , one was snap frozen for protein analysis, the other was preserved in RNA later (Ambion Inc.) and has been archived for microarray and Q-PCR analysis. These samples will provide intercomparison data for the different methods of environmental impact analysis in the final stage of the project.

 

STRIPED SEA BREAM

Fish were collected from 4stations, one at the mouth of the Kishon River within Haifa harbour which is heavily polluted and others south of Haifa bay, however, the longshore drift may mean that these sites are not pristine.

Sampling stations:

Pollution gradient 1 > 4.

RT PCR analyses sites 1 and 4.