Proteomics has been used in the clam Chamaelea gallina as a preliminary screening of changes in protein expression caused by pollutants, potentially useful as new biomarkers. Clams were exposed in water for seven days to four model contaminants, Aroclor 1254, copper(II), tributyltin (TBT), and arsenic(III), and cytosolic fractions were initially analyzed by two-dimensional (2-D) electrophoresis in 7 cm IPG strips (pH 4-7). On average, about 1000 spots were resolved and altered expression was qualitatively detected in 9-26 spots per treatment. Aroclor 1254, Cu(II) and As(III) had a mainly upregulating effect, in contrast to TBT. Altered protein expression was confirmed in 18 cm gels (at narrow pH ranges). The 15 spots more drastically altered were excised and analyzed by mass spectrometry (MS), and four proteins were identified. Aroclor 1254 and Cu(II) upregulated putative isoforms of tropomyosin and light chain of myosin. Actin was downregulated by Aroclor and Cu(II) but upregulated by TBT and As(III), while the opposite behavior was shown by a truncated actin form, homologous to the Drosophila act87E gene product. The exclusive identification of cytoskeletal proteins could reflect their relative abundance, their prevalence in databases in molluscs, or their role as major targets of pollutant-related oxidative stress.

In this study we have investigated protein changes in plasma of juvenile Atlantic cod (Gadus morhua) induced by crude North Sea oil and North Sea oil spiked with alkyl phenols and polycyclic aromatic hydrocarbons, a surrogate produced water composition. Using a proteomic approach, we identified 137 differentially expressed proteins at different levels of crude oil exposure. Many of the induced protein changes occurred at low levels of exposure. The results obtained with protein expression profiles after exposure to oil and surrogate produced water indicate effects on fibrinolysis and the complement cascade, the immune system, fertility-linked proteins, bone resorption, fatty acid metabolism as well as increased oxidative stress, impaired cell mobility and increased levels of proteins associated with apoptosis. Although the number of individuals and samples in this study is limited within each treatment group, the protein changes observed in this study represent a first screening for potential biomarker candidates in cod plasma reflecting potential effects of crude oil and produced water exposure on fish.

Produced water (PW) contains numerous toxic compounds of natural origin, such as dispersed oil, metals, alkylphenols (APs), and polycyclic aromatic hydrocarbons (PAHs). In addition, PW also contains many different chemicals which have been added during the oil production process. In the study described here, cod were exposed to real PW collected from an oil production platform in the North Sea. This was done in order to best recreate the most realistic field-exposure regime in which fish will be affected by a wide range of chemicals. The biological effects found in this study therefore cannot be assigned to one group of chemicals alone, but are the result of exposure to the complex chemical mixture found in real PW. Since APs are well known to cause endocrine disruption in marine organisms, we focused our chemical analysis on APs in an attempt to better understand the long-term effects of APs from PW on the biology of fish. In this study, cod were exposed to several concentrations of real PW and 17β-oestradiol (E(2)), a natural oestrogen, at different developmental stages. Cod were exposed to PW either during the embryo and early larvae stage (up to 3 months of age) or during the early juvenile stage (from 3 to 6 months of age). Results showed that, in general, APs bioconcentrate in fish tissue in a dose and developmental stage dependent manner during PW exposure. However, juveniles appeared able to effectively metabolise the short chain APs. Importantly, PW exposure had no effect on embryo survival or hatching success. However, 1% PW clearly interfered with the development of normal larval pigmentation. After hatching most of the larvae exposed to 1% PW failed to begin feeding and died of starvation. This inability to feed may be linked to the increased incidence of jaw deformities seen in these larvae. In addition, cod exposed to 1% PW, had significantly higher levels of the biomarkers vitellogenin and CYP1A in plasma and liver, respectively. No similar effects were seen in cod exposed to either 0.1% or 0.01% PW.

Primary cultures of salmon (Salmo salar L.) hepatocytes were analysed using (35)s-methionine/cysteine incorporation and SDS-PAGE gel electrophoresis (1 and 2-D) and Western blotting after treatment with representative environmental pollutants (benzo(a)pyrene (BaP), 2,3,3', 4,4'-pentachlorobiphenyl (PCB-105)1 arsenite (AsO2-) and cadmium (Cd)). The results demonstrated striking similarities in changes in protein expression after treatment with the different pollutants. Hsp70 (Hsp72/73) proteins were induced after treatment with all the compounds as shown by (35)S-methionine/cysteine labelling. However, high background levels of these proteins were shown with Western blotting and an anti-Hsp70 antibody, indicating a slow turnover of these proteins. The Hsp70s in salmon hepatocytes were extremely susceptible to degradation in urea used in 2-D electrophoresis, resulting in peptide fragments of 45-46 kDa. In addition to these Hsp70 fragments, arsenite induced several proteins of 42,38, and in the 30-32 kDa range. CYPlA (58 kDa) and an unidentified protein of 16 kDa were furthermore induced after treatment with the organic xenobiotics (BaP, PCB and the model compound β-naphthoflavone, BNF). CYPlA was expressed in a dose-dependent manner, and was resolved into several protein spots in 2-D Western blotting. Elevated levels of metallothionein and haem oxygenase (HO) were indicated in Western blots after treatment with cadmium or arsenite (only HO). The hepatocytes showed cytoplasmic protrusions after treatment with 35 μM arsenite and 100 μM Cd, indicative of cells entering apoptosis.